Protein-polysaccharides of pig laryngeal cartilage

1. Protein-polysaccharides of chondroitin 4-sulphate were extracted with neutral calcium chloride from pig laryngeal cartilage that was not completely homogenized. The protein-polysaccharides were purified by precipitation with 9-aminoacridine. On zone electrophoresis in compressed glass fibre at pH7.2 it was separated into two fractions, although two distinct zones were not obtained. These fractions, which had already been shown to differ in their antigenic determinants, also differed considerably in amino acid composition, total protein, hexose and glucosamine contents. 2. The fraction of higher mobility contained approx. 2% of protein and only traces of glucosamine. Serine and glycine accounted for over half the total amino acid residues, but aromatic, basic and sulphur-containing amino acids were not detected. The weight-average molecular weight, determined by sedimentation, was 230000. 3. Assuming that there was the same sequence of neutral sugars at the linkage points as in PP-L fraction (protein-polysaccharide light fraction), the approximate molar ratio of hexose to serine suggested that most of the serine residues were linked to chondroitin sulphate chains. Support for this was derived from the agreement between the weight-average molecular weight of the chondroitin sulphate-peptide after proteolysis, and the chain weight calculated from its serine content. The chain weight based on the serine content of the fraction of higher electrophoretic mobility was approximately similar. 4. In contrast, the fraction of lower electrophoretic mobility resembled PP-L fraction in its amino acid composition, protein and glucosamine contents. The presence of glucosamine, together with the higher hexose content, suggested that this fraction contained some keratan sulphate. 5. The relatively low molecular weight of the fraction of higher mobility enabled it to be extracted without complete disintegration of the cartilage. The unlikelihood of its being produced by autolytic enzymes is discussed.     

Laminin-like antigen in rat CNS neurons: distribution and changes upon brain injury and nerve growth factor treatment

Using several antibodies against rat or human laminin and an avidin-biotin immunocytochemical protocol, laminin-like immunoreactivity was detectable in the rat nervous system in expected locations, i.e., associated with blood vessels and reactive astrocytes. However, laminin staining was also abundantly present within neuronal cell bodies in most parts of the developing and adult rat CNS. Medial septum neuronal immunoreactivity was lost after septo-hippocampal disconnection, but could be preserved or even restored by intraventricular administration of nerve growth factor. Thus, at least for medial septum neurons, this laminin-like molecule can be accumulated or produced independent of direct hippocampal (target) contact. It remains to be determined whether CNS neuronal "laminin" processes activities similar to those found for laminin in vitro.     

Schwannoma cell-derived inhibitor of the neurite-promoting activity of laminin

During the purification of laminin-proteoglycan complexes from rat RN22 Schwannoma cell-conditioned medium, a laminin-rich fraction was obtained which lacked neurite-promoting activity. Since laminin from several sources is known to have potent neurite-promoting activity, this result suggested that either this laminin was inactive or its activity was somehow masked by associated molecule(s). The latter possibility was supported by the demonstration that the inactive laminin-containing fraction inhibited active laminin-containing fractions. This inhibitory activity was partially purified by using ion exchange chromatography and isopycnic centrifugation. The purified material contained proteoglycan based on its high affinity for cationic resin, high buoyant density, large heterodisperse appearance on electrophoretic gels, ability to label with inorganic sulfate, sensitivity to trypsin and glycosaminoglycan lyases, and heat stability. A quantitative in vitro bioassay was used to monitor the inhibitor after treatments aimed at defining its activity. The isolated Schwannoma-derived inhibitor (a) inhibits the neurite-promoting activity of purified rat, mouse, and human laminin; (b) is active whether presented to laminin in solution or after either the inhibitor or laminin is first bound to the culture substratum; (c) does not act by displacing laminin from the substratum; (d) can be prevented from binding to neurite-promoting laminin substrates by polyclonal and monoclonal anti-laminin or polyclonal anti-entactin antibodies; and (e) is abolished by proteases or glycosaminoglycan lyases but not by heat. The above results suggest that the neurite-promoting activity of laminin is subject to regulation through association with a proteoglycan and entactin.     

Metalloproteinase-Dependent Neurite Outgrowth within a Synthetic Extracellular Matrix Is Induced by Nerve Growth Factor

In order to assess the requirement for matrix metalloproteinases in neuronal regeneration, in vitro neurite outgrowth by chick dorsal root ganglionic neurons (DRGn) was examined within a reconstituted extracellular matrix. For these studies, cultured neurons were treated with a synthetic peptide inhibitor of metalloproteinases (spIMP), LMHKPRCGYPDVGG.spIMP inhibited all neuronal metalloproteinase activities in zymography and substrate-release assays and was used to examine the role of metalloproteinases in neurite outgrowth by DRGn. Cultures of dissociated DRGn rapidly extended neurites on planar extracellular matrix substrates and this rate of outgrowth was not affected by adding NGF or spIMP. In contrast, neurite extension within a three-dimensional gel of extracellular matrix increased nearly threefold after adding NGF. The NGF-induced neurite penetration was negated in the presence of spIMP but not by control peptide. Similar results were obtained using explanted dorsal root ganglia. These findings suggested that NGF-induced neurite outgrowth within an extracellular matrix involves metalloproteinase activity. Zymographic analysis of media conditioned by NGF-treated DRGn revealed a pair of gelatinolytic bands with apparent molecular masses 72 and 66 kDa, which comigrated as a single 66-kDa band after activation with an organomercurial agent. The gelatinase activities were calcium- and zinc-dependent and were absent from zymograms developed in the presence of spIMP, indicating that NGF-treated DRGn release and activate a 72-kDa metalloproteinase. Samples from DRGn cultures treated with low levels of NGF contained similar amounts of latent and activated metalloproteinase, while high levels of NGF induced an apparent increase in total metalloproteinase secretion and a substantially greater proportion of activated enzyme. Western blot analysis showed this metalloproteinase was immunologically similar to 72-kDa type IV collagenase and immunoassays revealed that this matrix metalloproteinase was increased threefold by high NGF. Furthermore, after high NGF treatment, DRGn media contained sixfold more metalloproteinase activity in assays of matrix degradation. In summary, these results indicate that NGF enhanced metalloproteinase-dependent neurite outgrowth of DRGn within a reconstituted extracellular matrix. Also, NGF increased the expression and activation of 72-kDa type IV collagenase, suggesting a role for this matrix-degrading metalloproteinase in neuronal regeneration.     

Stability of bilirubin oxidase in organic solvent media: a comparative study on two low-water systems

The storage stability of bilirubin oxidase was studied in water-in-oil CTAB microemulsions with a chloroformrich continuous organic phase. The kinetics of the inactivation process were best described by a double exponential equation. Approximately half of enzymatic activity was lost during a "fast" phase with a half life of ca. 50 min, whereas the remaining activity was lost much more slowly (half life ca. 1000 min). Rates of inactivation were not affected significantly by variation of either solvent composition or concentration of water droplets, but inactivation was more rapid when droplet size was very small. Steady-state enzyme kinetics were studied at various stages in the inactivation process, and it was shown that inactivation occurred without change in the K(m) of the enzyme for bilirubin. Stability was also studied in a liquid/solid two-phase system; it was found that the inactivation process in this system; it was found that the inactivation process in this system was best described by a single exponential term. The rate was similar to the "fast" phase rate observed in the water-in-oil microemulsion system. Inactivation of the enzyme slow. Addition of the surfactant CTAB to the aqueous environment increased the rate of inactivation to levels comparable to those of the "slow" phase observed in water-in-oil microemulsions. (c) 1993 Wiley & Sons, Inc.     

Leukocyte kinetics in the human lung: role of exercise and catecholamines

In six normal supine subjects epinephrine infusion produced a greater leukocytosis with smaller changes in heart rate and blood pressure than did norepinephrine or isoproterenol. Upright exercise in those subjects produced a greater leukocytosis than supine exercise at the same work load. To determine the lung's participation in these events, indium-labeled neutrophils (PMN) were given to four of the subjects. We found that 20-25% were retained in the first pass through the lung when compared with technetium-labeled erythrocytes. The number of labeled PMN in the lung gradually decreased and the number in the spleen and the liver increased. Exercise and catecholamine infusion caused an acceleration in the release of labeled cells from the lung, an increase in both labeled and unlabeled cells in the peripheral blood, and an increase in the number of labeled cells in the liver and spleen. This suggests that increased perfusion of low-flow areas in the lung may contribute to the increased leukocytosis seen in association with both exercise and catecholamine infusion.     

An automated version of the periodate-thiobarbituric acid assay for analysis of delta-4,5 unsaturated uronic acids and its application to the assay of hyaluronic acids and chondroitin sulfates

An automated periodate-thiobarbituric acid assay of Δ-4,5 unsaturated uronic acids which avoids extraction of chromogen has been developed and applied to the analysis of hyaluronic acid and chondroitin sulfates in standard glycosaminoglycan mixtures and in biological samples following digestion with eliminase enzymes. Assay of hyaluronic acid was linear between 0.1 and 2.5 μg of uronic acid, when digested with hyaluronidase from S. hyalurolyticus and use directly in the automated procedure. The measurement of unsaturated disaccharide standards (25–100 μm) derived from chondroitin sulfates was also linear although the color yields were different. The proportions of chondroitin sulfate isomers were estimated by assay of the unsaturated chondroitin disaccharides which has been separated by thin-layer chromatography.     

Effects of pirbuterol and sodium nitroprusside on pulmonary haemodynamics in hypoxic cor pulmonale.

The acute haemodynamic effects of oral pirbuterol (a beta-agonist) were contrasted with those of sodium nitroprusside, a vasodilator, in six patients with hypoxic chronic bronchitis and emphysema. Sodium nitroprusside (1-5 mg/kg intravenously) reduced mean pulmonary arterial pressure and total pulmonary vascular resistance significantly (p less than 0.01) without change in cardiac output or right ventricular ejection fraction, measured by radionuclide ventriculography. Oral pirbuterol (22.5 mg) produced a greater reduction in total pulmonary vascular resistance than sodium nitroprusside, largely as a result of increasing cardiac output. Right ventricular ejection fraction also increased significantly after pirbuterol (p less than 0.01). Pirbuterol in a lower dosage (15 mg by mouth) in six further patients with hypoxic chronic bronchitis and emphysema produced similar changes in total pulmonary vascular resistance and right ventricular ejection fraction. Nine of the patients who were studied acutely thereafter received pirbuterol 15 mg thrice daily for six weeks, which produced a significant fall in systolic pulmonary arterial pressure and a rise in right ventricular ejection fraction (p less than 0.01), without a significant fall in arterial oxygen tension. Pirbuterol acts as a vasodilator on the pulmonary circulation in these patients and may in addition improve right ventricular performance by an inotropic action.