The human colonic cell line PC/AA, derived from an adenoma, retains in vitro colonic cell differentiation, notably the production of mucus glycoproteins. The PC/AA adenoma cells produce an extracellular gel layer in culture. The PC/AA gel could be isolated by extraction of the cell cultures with guanidine hydrochloride. The extracted material was purified by gel filtration and caesium chloride density-gradient centrifugation and showed properties typical of mucus glycoproteins, namely, a carbohydrate content above 60% of dry weight rich in N-acetylgalactosamine and sialic acid and low in mannose; an amino acid composition with high serine threonine and proline content; a molecular weight above 1,000 kDa on Sepharose CL 4B chromatography and on SDS-polyacrylamide gel electrophoresis under reducing conditions (greater than 200 kDa); a buoyant density of approximately 1.48 g/ml and the release of oligosaccharides by the alkaline beta-elimination reaction. Comparison of the gel mucus glycoprotein purified from premalignant PC/AA cells with normal human colon mucin showed that it has a higher sialic acid content. This suggests that higher sialic acid levels may precede the development of malignancy.
Cultures were initiated from meristems (0.5 mm) of the rose cultivar Queen Elizabeth (floribunda) and from both shoot-tips and nodal explants (3-5 mm) of cultivars Sunburst Red, Toy Clown (miniatures) and Fiona (ground cover). Average proliferations of 5.0, 3.1, 1.3 and 2.5 shoots were obtained per culture cycle respectively on Murashige & Skoog (MS) medium with BA (1.0 mg 1-~ ), NAA (0.1 mg 1-1) and GA 3 (0.1 mg 1-1 ). With cv. Fiona, the proliferation rate was more than doubled by removal of the shoot apex. The rate of proliferation of cv. Queen Elizabeth was significantly increased by using long shoots (> 2 cm in length) and by re-culturing shoots to fresh medium every 3 weeks. In vitro rooting percentage with cv. Queen Elizabeth was enhanced by using long shoots ( > 2 cm) and by dilution of MS medium to 1/4 strength. Transfer of shoots for direct rooting in compost was significantly improved by pre-culturing shoots for two weeks in vitro in media containing IAA, and by the use of sorbarods.
From these results, it is clear that certain oligosaccharides are much better substrates than others for each a-mannosidase and that for each substrate, certain mannose residues are preferentially cleaved. Each enzyme degrades the oligosaccharides by ii specific pathway, which is governed by steric accessibility. resulting from the conformation o f the oligosaccharide a s it is hound by the enzyme.T h e substrate specificities for the human, hovine. and feline lysosomal u-mannosidases. together with the absence in animals other than humans and rodents of lysosomal endo-N-acetyl-~-~-glucosaminidase [ 1 I], offer some explanation of the patterns of oligosaccharides accumulated in tissues and excreted in urines of humans and animals with a-mannosidosis [ 1-3. 121. T h e resistance of the core a( 1 -(,)-linkage t o the major human lysosomal u-mannosidase supports the hypothesis that the hydrolysis of this linkage is catalysed by another activity [ 131. Similarly, the facile hydrolysis of the core a( 1 -3)-linkagc by the human enzyme is consistent with the presence o f this linkage in the major urinary oligosaccharide in the absence of the enzyme.T h e well-dcfined specificities for oligosaccharide structures suggest parallels between the catabolic enzymes. and the a-mannosidases involved in the processing of N-linked glycans j 8. 101. This investigationWIS supported h y grant HI>-? I OX7 f r o m the
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