Nicola Di Ferrante

Deficiencies of Glucosamine-6-Sulfate or Galactosamine-6-Sulfate Sulfatases Are Responsible for Different Mucopolysaccharidoses

[1-3H]Galactitol-6-sulfate, N-[1-3H]acetylgalactosaminitol-6-sulfate, N-[1-3H]acetylglucosaminitol-6-sulfate, N-acetylglucosamine-6-sulfate, and 6-sulfated tetrasaccharides from chondroitin-6-sulfate have been used for the measurement of 6-sulfatase activity of extracts of normal skin fibroblasts and of fibroblasts cultured from patients with genetic mucopolysaccharidoses. With these substrates, extracts of fibroblasts derived from Morquio patients lack or have greatly reduced activities for galactitol-6-sulfate, N-acetylgalactosaminitol-6-sulfate, and 6-sulfated tetrasaccharides but have normal activity for N-acetylglucosamine-6-sulfate and its alditol; those derived from a patient with a newly discovered mucopolysaccharidosis have greatly reduced activity for N-acetylglucosamine-6-sulfate and its alditol but normal activity for galactitol-6-sulfate, N-acetylgalactosaminitol-6-sulfate, and the 6-sulfated tetrasaccharides. These findings demonstrate the existence of two different hexosamine-6-sulfate sulfatases, specific for the glucose or galactose configuration of their substrates. Their respective deficiencies, causing inability to degrade keratan sulfate and heparan sulfate in one case and keratan sulfate and chondroitin-6-sulfate in the other, are responsible for different clinical phenotypes.

High-speed gel-permeation chromatography of glycosaminoglycans: its application to the analysis of heparan sulfate of embryonic carcinoma and its degradation products by tumor cell-derived heparanase.

A high-speed gel-permeation chromatographic system for analyzing glycosaminoglycans which uses two 0.7 X 75-cm stainless-steel columns containing Fractogel (Toyopearl) TSK HW-55(S), was developed. Glycosaminoglycans were applied and eluted with a 0.2 M sodium chloride solution and monitored by ultraviolet absorption at 210 nm or radioactivity. The best resolution of glycans was obtained at 55 degrees C at a flow rate of 1.0 ml/min. Acidic and neutral glycans in the molecular weight (Mr) range 600-60,000 eluted within 45 min. A linear relationship was found between retention time and molecular weight using standard glycosaminoglycans, chitin oligosaccharides, and a porcine thyroglobulin glycoprotide. This system was used to analyze the heparan sulfate synthesized by PYS-2 embryonic carcinoma cells and the degradation products produced by incubating it with extracted glycosidases from metastatic B16 melanoma cells. The results indicated that B16 melanoma cells contain at least two different heparan sulfate degradative activities, one of which appears to be an endoglycosidase.

The binding of chondroitin 6-sulfate to plasma low density lipoprotein

The interaction in vitro of several sulfated glycosaminoglycans with low density lipoproteins (LDL) has been studied. Chondroitin 6-sulfate and heparin were the only ones to produce turbidity when added to LDL in presence of Ca2+. However, when these two glycosaminoglycans were applied to LDL-affinity columns in presence of Ca2+, only chondroitin 6-sulfate was retained. Partially desulfated chondroitin 6-sulfate was not retained on LDL-affinity column, indicating the relevance of sulfate groups in the binding of LDL. Since chondroitin 4-sulfate and heparin, with a sulfate content respectively equal to and greater than that of chondroitin 6-sulfate, are not retained on LDL-affinity columns, the factors relevant to the binding of LDL are probably the conformation of the glycan in solution and the orientation of its sulfate groups.

Collagen Breakdown and Nitrogen Dioxide Inhalation

Measurements of urinary hydroxylysine glycosides indicate that considerable collagen degradation occurred during the reentry into the earths atmosphere of the American astronauts of the Apollo-Soyuz mission. Since the crew accidentally inhaled nitrogen dioxide, a recognized pulmonary irritant, and showed clinical and roentgenographic signs of diffuse chemical pneumonitis, it is likely that collagen degradation occurred in the pulmonary parenchyma.

Sensitive methods for the determination of ester sulfate in biological systems.

Abstract The benzidine and rhodizonate methods for the assay of inorganic sulfate have been modified in order to increase their sensitivity, rapidity, and safety. With these modifications, amounts of inorganic sulfate ranging between 0.15 and 15 μg (1.56 to 156 nmoles) may be conveniently measured. At this level of sensitivity, the methods may be used for the assay of several sulfohydrolases of biological relevance without using 35SO4-labeled substrates.

A method for α-L-iduronidase assay

It is now established that a-L-iduronidase is the lysosomal hydrolase whose deficiency is responsible for the pathogenesis of Hurler and Scheie diseases [l-3]. The availability of the synthetic substrate, phenlycY-Liduronide [4] , has made possible to assay the levels of this enzyme in homogenates of various organs [5], cultured skin fibroblasts and amniotic fluids cells [61The methods used for the measurement of cu-Liduronidase detect the released phenol with the FolinCiocalteau-carbonate reagent. The interference of this protein has been reduced in various ways: by precipitating first some of the protein with the FolinCiocalteau reagent and adding the alkali to an aliquot of the clear supernatant [6] ; or by extracting the released phenol in toluene, re-extracting it in an alkaline aqueous solution and performing the color reaction on the latter [5] . The necessity of working with low substrate concentrations and minimal volumes, the inefficient precipitation of protein with the Folin-Ciocalteau reagent, the need for microglassware, repeated extractions and occasional clarification of cloudy extracts, are factors which limit the usefulness of these methods when low levels of activity have to be measured in biological fluids or tissue extracts having a high protein content. The method proposed here is based on the reaction of N,2,6-trichloro-p-benzoquinoneimine (TCBQ) with phenol, to form phenolindophenol and, upon alkalinization, a blue chromogen absorbing at 6 10

Collagen breakdown and ammonia inhalation.

Measurement of several urinary metabolites of hydroxylysine indicates that considerable collagen degradation occurred in four individuals immediately after they had inhaled concentrated ammonia vapors. Since clinical and/or radiological evidence of intense upper respiratory and pulmonary inflammation were evident, it is likely that collagen degradation occurred at the level of the respiratory system.

Immediate endocrine and metabolic consequences of traumatic quadriplegia in a young woman.

Onset of paralysis by cervical spinal cord injury led immediately to temporary adrenocortical activation and, within 2 days, to sustained skin and bone breakdown. Urine cAMP was increased, blood parathyroid hormone, renin activity, and electrolytes were normal, and fluid and electrolytes balance became negative during the initial 6 days of paralysis.

Plasma glycosaminoglycans in normal individuals of various age.

Abstract Our previous studies have demonstrated that the high-sulfated glycosaminoglycans (GAG) of human plasma interact with low-density and high-density plasma lipoproteins (LDL and HDL), while the more abundant, low-sulfated plasma GAG prevent such interaction. Therefore, we have suggested that the ratio of these two species of circulating GAG might affect the rheological properties and metabolic fate of plasma lipoproteins. In this study the high-sulfated and low-sulfated plasma GAG have been measured in 79 normal individuals of both sexes, belonging to increasing age groups: 20–29, 30–39, 40–49 and 50–59 years old. No significant differences were found between males and females of the same group. No significant differences were found between the levels of high-sulfated and low-sulfated plasma GAG of the first and second group of individuals. The average values found and their ratios were in agreement with those reported previously for the same age groups. The average values of high-sulfated and low-sulfated plasma GAG of the third and fourth group of individuals were not statistically different but they were significantly higher than those of the first two groups. Moreover, the ratio high-sulfated/low-sulfated plasma GAG in older individuals was between 0.41 and 0.45, as compared to values of 0.27–0.37 found in the younger individuals. This increased ratio reflects a greater increase in the level of the high-sulfated species which, by interacting with circulating lipoproteins, might cause physical and metabolic modifications favoring their deposition and permanence within the arterial wall.

Antigenic determinants of two components of proteoglycan complex from bovine cartilage

The immunological properties of a glycoprotein fraction and of proteoglycan subunits obtained from bovine nasal cartilage by nondisruptive methods of isolation have been studied. Using the techniques of hemagglutination and hemagglutination inhibition, we found that the glycoprotein contains most of the species‐specific determinants, whereas the proteoglycan subunits contain most of the cross‐reacting ones.