Richard H. Pearce

The structure and degradation of aggrecan in human intervertebral disc

The ability of the intervertebral disc to resist compression is dependent on its high proteoglycan concentration. The disc proteoglycans are classified as aggregating or non-aggregating depending on their ability to interact with hyaluronan. The majority of the aggregating proteoglycans are derived from aggrecan, though their glycosaminoglycan substitution pattern has not been determined. In contrast, the origin of the non-aggregating proteoglycans is unclear, though it has been postulated that they are derived from aggrecan by proteolysis. The present work demonstrates that keratan sulfate (KS) in the glycosaminoglycan-binding region of disc aggrecan is confined to the KS-rich domain of the core protein and is not present in association with chondroitin sulfate (CS) in the CS1 and CS2 domains. It also shows that the non-aggregating disc proteoglycans are derived from aggrecan, with the large molecules possessing both the KS-rich and CS1 domains and the smaller molecules being generated from either the KS-rich or CS2 domain. The origin and spectrum of disc proteoglycan heterogeneity is the same in both the annulus fibrosus and nucleus pulposus.

Analysis of hyaluronic acid in the diagnosis of malignant mesothelioma

Using a modified papain digestion cetylpyridinium salt precipitation method, glycosaminoglycans were isolated from 21 mesotheliomas, 34 primary lung carcinomas, 12 carcinomas of other sites, and 7 soft tissue sarcomas. Qualitatively, hyaluronic acid (HA) was present in 20 of 21 mesotheliomas, about half of the primary lung adenocarcinomas, and all of the soft tissue sarcomas. On the average, HA constituted 45% of the total glycosaminoglycans in the mesotheliomas and 28% of the total in the lung cancers. Quantitatively, mesotheliomas contained statistically greater amounts (mean value, 0.74 mg/g) of HA than primary lung adenocarcinomas (mean value, 0.08 mg/g), but were not statistically different from soft tissue sarcomas (mean value, 2.01 mg/g) or primary ovarian serous neoplasms (mean value, 0.92 mg/g). The study concludes that, contrary to previous reports, HA is neither the sole nor the predominant glycosaminoglycan in most mesotheliomas, but, given the proper clinical and histologic setting, the finding of sufficiently high levels (greater than 0.4 mg/g dry tissue extract) supports the diagnosis of mesothelioma when the alternative diagnosis is primary adenocarcinoma of lung.

Concentration of plasma albumin in its accessible space in postmortem human dermis

This study was designed to measure the effective concentration of plasma albumin in the interstitial space of human dermis. Discs of tissue taken postmortem from four donors have been separately analyzed for their content of plasma albumin and equilibrated with 125I-labeled monomeric plasma albumin in a specially designed cell which limited tissue swelling. The equilibrated discs and their surrounding fluid were assayed for radioactivity and the tissue space accessible to albumin was calculated after correction for swelling. The albumin content of serum was also measured. The concentration of albumin in the accessible space of the tissue ranged from 0.45 to 0.93 that in serum, averaging 0.68. The fraction of the total interstitial fluid accessible to albumin averaged, for three normal dermises, 0.35 and for an overhydrated specimen, 0.51. Thus, the effect of volume exclusion should be considered in measurements of the concentrations of plasma proteins in tissue.

Fractionation of anionic glycosaminoglycans by ion-exchange chromatography

1. 1. The ion-exchange properties of Dowex 1-X2 (Cl−), 100–200 mesh, have been studied for the common anionic glycosaminoglycans found in animal tissues: capacity, range of salt concentrations for elution, and recovery. 2. 2. 8 M urea influences the resin-glycosaminoglycan bond. 3. 3. DEAE and ECTEOLA resins give inferior results. 4. 4. The elution profile depends to a greater extent on the support and conditions of elution than on the properties of the glycosaminoglycan. 5. 5. Examples of several applications are given.

A Model of Unloaded Human Intervertebral Disk Based on NMR Relaxation

NMR relaxation rates were related to the composition of the nucleus pulposus from 11 and anulus fibrosus from six human intervertebral disks. Tissue water was proportional to glycosaminoglycan (GAG) and residue, the noncollagen, non‐GAG portion of the dry weight (R2= 0.74). The solid signal fraction depended on collagen and residue protons (R2= 0.89). 1/T1 was proportional to collagen and residue (R2= 0.97). T2 showed 2–4 components labeled A, B, C, and D, with means ± standard deviations of 3.1 ± 1.6, 17.5 ± 9.5, 64 ± 22, and 347 ± 162 msec. Signal fractions of A and B depended on the collagen‐associated water protons (R2= 0.94 and 0.85), C on residue‐associated water protons (R2= 0.82), and D on GAG‐associated water protons (R2= 0.74). The data led to a model of disk architecture in which the collagen and residue were largely solid, forming distinct water compartments; the remaining water was present in a proteoglycan gel. Magn Reson Med 43:34–44, 2000.

Fractionation of rat cutaneous glycosaminoglycans using an anion-exchange resin

Abstract 1. 1. In the presence of dilute acetic acid, chondroitin 4-sulfate can be eluted from AG 1 anion-exchange resin separately from dermatan sulfate. 2. 2. This principle has been used as the basis of a simple method for the separation of the bulk of the hyaluronate and dermatan sulfate from the other glycosaminoglycans of rat skin. 3. 3. The cutaneous glycosaminoglycans behave differently than reference glycosaminoglycans in this system.

Assay of keratan sulfate as anion-Exchanger bound hexose

An assay for keratan sulfate in papain digests of human intervertebral disc and other tissues has been developed. The digest is applied to the acetate form of a tertiary amine acrylic anion-exchange resin, the oligosaccharide hexose is removed by washing the resin with 0.2 M sodium acetate buffer pH 5.0, then the keratan sulfate is eluted quantitatively with 1.0 M pyridinium sulfate pH 2.5 and assayed for hexose by the anthrone reaction. The keratan sulfate content of human intervertebral disc tissues ranged from 7 to 78 mumole galactose equivalents/g fresh weight; the root mean square error was 2 mumole/g; 10-25 mg of tissue were required. The separation of oligosaccharides from keratan sulfate was confirmed by gel permeation chromatography, sugar composition, ester sulfate analysis, and nuclear magnetic resonance.