Jane R. Scocca

An accelerated system for analysis of neutral sugars in complex carbohydrates.

Abstract An accelerated ion-exchange chromatography system for analysis of neutral sugar components in complex carbohydrates is described. Using DA-4 anion-exchange resin, and gradient elution with sodium borate, only 2.5 hr is required per analysis in this system.

Rapid automatic analysis of sugar components in glycoproteins: I. Hexosamines☆

Abstract A rapid method is described for the automatic determination of hexosamines in glycoproteins. Hexosamines are separated by cation-exchange chromatography and automatically determined by a neocuproine reducing sugar method. The method is highly reproducible and as little as 1 μg of glucosamine can be measured accurately.

Alterations in cultured fibroblasts of sibs with an infantile form of a free (Unbound) sialic acid storage disorder

Summary: Cultured fibroblasts from two sibs with generalized hypertonia, hepatosplenomegaly, and psychomotor retardation within the first year of life were found to have unusual morphologic features. When examined by phase microscopy, the unstained and unfixed cells contained a large number of vacuolated structures whose gross appearance resembled that of a honeycomb in the cell cytoplasm. Electron microscopy studies, following fixation, showed the “honeycombing” to be the result of numerous, closely packed, cytoplasmic, membrane-bound vacuoles. In some of these structures the remains of fibrillogranular material could be detected.Biochemical analysis of crude sonicates of these cells revealed increased levels (4–7 × N) of an acid soluble component that reacted with thiobarbituric acid. Analysis of trimethylsilyl derivatives of this material by gas liquid chromatography and mass spectrometry showed it to be indistinguishable from sialic acid (N-acetylneuraminic acid).Quantitation of this material from the cells of one of the sibs after isolation on a Dowex column yielded 39.8 nmoles of free (unbound) sialic acid per mg protein whereas normal fibroblasts had 1–2 nmoles per mg. Bound sialic acid levels were at the upper limits of normal (24.8 versus 11–23 nmoles per mg protein). The concentration of cytidine monophosphate-sialic acid was normal.After incubation of the patients fibroblasts with [3H]-N-acetyl-mannosamine for 72 h, there was a 7-fold increase (compared to normal fibroblasts) in the amount of radioactivity in free sialic acid present in the acid soluble fraction. The amount of labeled, bound sialic acid in the acid-insoluble pool, however, was the same in both patient and control fibroblasts.

Chinese hamster ovary cells with reduced hexokinase activity maintain normal GDP‐mannose levels

Parental Chinese hamster ovary (CHO) cells were mutagenized and subjected first to a mannose suicide selection technique and second to a screen of individual colonies grown on polyester discs for reduced mannose incorporation into protein. The incorporation of radioactivity for the selection and the screen was conducted at 41.5°C instead of the normal growth temperature of 34°C in order to allow for the isolation of temperature‐sensitive lesions. This selection/screening procedure resulted in the isolation of MI5–4 cells, which had three‐ to five‐fold lower incorporation of [2‐3H]mannose into mannose 6‐phosphate, mannose 1‐phosphate, GDP‐mannose, oligosaccharide‐lipid, and glycoprotein at 41.5°C. We detected no difference in the qualitative pattern of mannose‐labeled lipid‐linked oligosaccharides compared to parental cells. MI5–4 cells synthesized dolichol. The defect of MI5–4 cells was determined to be in hexokinase activity; crude cytosolic extracts were eight‐ to nine‐fold lower in hexokinase activity in MI5–4 cells compared to parental cells. As a result of this defect, incorporation of labeled mannose from the medium was significantly decreased. However, the level of GDP‐mannose in MI5–4 cells was 70% of normal. The phenotype of MI5–4 was a lower specific activity of labeled GDP‐mannose, not a substantial reduction in the level of GDP‐mannose. Consistent with these results, no alterations in the glycosylation of a model glycoprotein, G protein of vesicular stomatitis virus, were observed. These cells grew slower than parental cells, especially in low‐glucose medium. J. Cell. Biochem. 72:56–66, 1999.

Evidence of gene amplification in tunicamycin-resistant chinese hamster ovary cells

Restriction digests of genomic DNA from tunicamycin-resistant Chinese hamster ovary cells, 3E11, were probed with the yeast transferase gene, ALG7. The data presented suggest moderate amplification of the N-acetylglucosaminyl-1-phosphate transferase gene occurred in these cells, consistent with the previously observed chromosomal translocations and increased enzymatic activity. This is the first example of gene amplification as a mechanism for aberrations in N-linked glycosylation.

Chromatography of methyl ethers of d-glucosamine

Abstract Identification of methyl ethers obtained by methylation and subsequent hydrolysis is a powerful technique for determination of linkage positions in structure studies of complex carbohydrates. Although methyl ethers of neutral sugars have been separated by various chromatographic methods, separation of methyl ethers of 2-amino-2-deoxy- d -glucose has been more difficult. Only recently, successful procedures for separation of methyl ethers of d -glucosamine based on thin-layer (1), gas (2), and column (3) chromatography have appeared in literature. We wish to report here a method for separation of d -glucosamine methyl ethers using a combination of partition and ion-exchange chromatography.

Evidence for non-lysosomal storage of N-acetylneuraminic acid (sialic acid) in sialuria fibroblasts.

The results of the investigations reported here indicate that patients affected with the infantile sialic acid storage disorder (ISSD) and the original French sialuria patient suffer from distinct and fundamentally different disorders. While phase microscopy and immunochcmical studies demonstrated abnormal storage within intracellular inclusions in ISSD cells, no morphological evidence of storage within any subcellular organelles was found in the sialuria cells. Moreover, comparative subcellular fractionation studies on gradients of colloidal silica showed the excess sialic acid in ISSD cells to be located within the light (buoyant) lysosomal fraction, while the excessive, free sialic acid in the sialuria cells was found in the cytoplasmic fraction with no increased storage within the lysosomal tractions. It is concluded that the sialic acid abnormalities in ISSD and the French type of sialuria are the result of very different biochemical and genetically unrelated abnormalities.