Phyllis M. Sampson

PERMEABILITY OF PULMONARY ENDOTHELIUM TO NEUTRAL AND CHARGED MACROMOLECULES

Although the presence of negative charges on the surface of cells has been studied extensively, the importance of negative charges as determinants of capillary permeability is just beginning to be explored.1 Early studies on the reflection coefficient for anionic and uncharged dextrans in rabbit ear capillaries had shown that these vessels were less permeable to anionic than to neutral dextrans.2 However, the anatomic site for this charge restriction was not identified. In the renal glomerulus, the basement membrane constitutes more of a barrier to the passage of anionic macromolecules than to their neutral or cationic counterparts.3 In contrast to the arrangement in the glomerulus where endothelial cells are provided with open fenestrae that allow direct contact between blood and basement membrane, the capillary endothelium of the lung forms a continuous layer separating blood from the basement membrane and the interstitium. In the present paper we have examined whether molecular charge influences the movement of macromolecules from plasma to lymph in lungs and the role of the capillary endothelium in mediating this effect. We found that the distribution of anionic sites on the surface of the capillary endothelium is not uniform. In contrast to skin and glomeruli, in the lung the movement of anionic molecules from plasma to lymph is enhanced compared to the movement of uncharged molecules of comparable size.

Detecting proteoglycans immobilized on positively charged nylon

Proteoglycans (PG) immobilized on positively charged Nylon 66 are detected readily by staining with Alcian blue. With the exception of hyaluronic acid, free glycosaminoglycans appear unreactive when treated similarly. Immobilization was performed by dot blotting or by electrophoretic transblotting from various gel supports. When transblotted to positively charged Nylon 66 from large-pore agarose-acrylamide gels, levels of 10-50 ng of PG could be detected by Alcian blue staining. This procedure appeared to be nearly 10(2) times more sensitive than staining of gels with toluidine blue. The transblot and staining procedure also appears to be effective with PG separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was applied to a preparation enriched in basement membrane components.

Detection of glycosaminoglycans at the one-nanogram level by 125I-cytochrome c.

The basic protein cytochrome c forms stable ionic complexes with all known glycosaminoglycans. When labeled with 125I, cytochrome c is capable of detecting exceptionally small quantities of glycosaminoglycans. Subsequent to electrophoresis on cellulose acetate strips using pyridine formate buffer at pH 3, followed by ethanol fixation, and treatment with 125I-cytochrome c, all the known glycosaminoglycans are detected at minimum levels of 1 ng/0.25-microliter application. The method can be used for quantification of glycosaminoglycans in other electrophoretic buffer systems also.

Isolation and partial characterization of proteoglycans from sheep lung parenchyma.

Greater than 90% of the proteoglycans of sheep lung parenchyma, as measured by uronic acid, were solubilized employing a sequential procedure with guanidine hydrochloride, dithiothreitol and Triton X-100. The amounts solubilized were 68.7%, 16.2% and 5.9%, respectively. The guanidine hydrochloride extract was chromatographed using DEAE-cellulose in urea and eluted with increasing concentrations of NaCl. A major fraction (containing a 6.5-fold enrichment of uronic acid) was obtained with 0.5 M NaCl and further purified by Sepharose Cl-6B chromatography in guanidine hydrochloride. To demonstrate the presence of protein-linked glycosaminoglycans, the void volume peak containing protein and uronic acid was digested with papain and rechromatographed. Evidence for the presence of proteoglycans was obtained by observing an almost complete loss of uronic acid in the void volume and the appearance of a uronic acid peak in the included volume, migrating in the same area as single-chain glycosaminoglycans. Electrophoretic migration and disappearance of bands in electrophoresis after digestion with specific mucopolysaccharide lyases indicated that the small amount of uronic acid remaining in the void volume was hyaluronic acid whereas the included volume contained hyaluronic acid, heparan sulfate, chondroitin sulfates and/or dermatan sulfate.

Glycosaminoglycans and Chondroitin/Dermatan Sulfate Proteoglycans in the Myocardium of a Non-Human Primate

Lyophilized mid-wall left ventricular myocardial tissues of the long-tailed non-human primate, Macaca fascicularis, were examined for the presence of glycosaminoglycans and chondroitin/dermatan sulfate proteoglycans. Mean uronic acid concentration in all samples was 0.97 +/- 0.27 micrograms per mg dry weight myocardium. The distribution of the glycosaminoglycans in the myocardium, determined by cellulose acetate strip electrophoresis was 62 +/- 4% heparan sulfate, 20 +/- 6% hyaluronan, and 16 +/- 5% chondroitin/dermatan sulfate. The analysis of chondroitin/dermatan sulfate proteoglycans, done directly on the extracts of lyophilized myocardium using agarose-acrylamide gel electrophoresis and Western blotting with monoclonal antibodies to various carbohydrate epitopes and with polyclonal antibodies to the protein core, showed the presence of biglycan and decorin. That these two and no other chondroitin/dermatan sulfates were present was established by core protein analysis using SDS PAGE and Western blotting. Quantification of chondroitin/dermatan sulfate proteoglycans uncovered high individual specific variability of the chondroitin/dermatan sulfate epitopes, but only moderate variability of biglycan and decorin core proteins. The variability of the chondroitin/dermatan sulfate epitopes is most likely related to individual specific differences in chain number, iduronate content and sulfation patterns of biglycan and decorin.

Detection by 125I-cationized cytochrome c of proteoglycans and glycosaminoglycans immobilized on unmodified and on positively charged Nylon 66

We have examined the detection by a 125I-labeled basic protein, cationized cytochrome c, of selected proteoglycans (PGs) and standard preparations of glycosaminoglycans (GAGs) immobilized on Nylon 66 and also on positively charged Nylon 66. Immobilization on Nylon 66 appears to allow a relative freedom of interaction between PGs or GAGs and 125I-cationized cytochrome c, but a more restricted reaction was observed when PGs and GAGs were immobilized to positively charged Nylon 66. On this support PGs with large numbers of GAG side chains reacted well with 125I-cationized cytochrome c, but GAGs were minimally reactive. By taking advantage of some of the properties of large-pore agarose-acrylamide gels, rapid partial characterization of some PGs can be accomplished in the 10-ng range, and therefore at a sensitivity equal to PGs with internal biosynthetic labels.

[19] Detection of glycosaminoglycans with 125I-labeled cytochrome c

Publisher Summary The mobility of individual glycosaminoglycans (GAG) on cellulose acetate strip electrophoresis in conjunction with selective hydrolysis by specific mucopolysaccharide lyases has been used for many years for qualitative and quantitative analyses of GAG. In this method, the GAG are visualized by ortho- and metachromatic dyes. This chapter discusses the methods of detection of glycosaminoglycans with I-Labeled cytochrome. Out of many available buffer electrophoretic systems, only three have been used for this purpose. Only two cellulose acetate strips have been used for the detection of glycosaminoglycans with I-Labeled cytochrome. The assay described in the chapter has the potential of being converted to fluorescent or enzyme-linked assays, but because cytochrome c has native “peroxidase activity,” coupling to an enzyme may not be necessary. The interaction of GAG with cytochrome c or other basic proteins offers new opportunities for detection of GAG that are not available when GAG are visualized by ortho- and metachromatic dyes.