Identification and partial characterization of two inducible gelatin-cleavage activities localized to the sea urchin extraembryonic matrix, the hyaline layer.

We have identified two inducible, gelatin-cleaving activities in the sea urchin extraembryonic matrix, the hyaline layer. Isolated hyaline layers, incubated in the presence of benzamidine, were devoid of gelatin-cleavage activities with apparent molecular mass less then 80k. However, when layers were incubated for 9-11 h in the absence of benzamidine, gelatin-cleavage activities, with apparent molecular mass 40- and 50k, were detected. Induction required the presence of NaCl and CaCl(2) at concentrations similar to those found in seawater and readdition of the reversible serine protease inhibitor benzamidine prevented induction. Both gelatin-cleaving activities were activated by calcium at a concentration similar to the calcium concentration found in seawater. Magnesium, also a major cationic species present in seawater, could not replace calcium as the activating ion. In addition, magnesium could not compete with calcium for binding to the gelatinases. Both cleavage activities showed substrate specificity and each failed to cleave bovine serum albumin, bovine hemoglobin or casein. Cleavage activity towards gelatin was inhibited by benzamidine and aminoethyl benzenesulfonyl fluoride, indicating that both activities belonged to the serine class of proteases. The induced 40-kDa activity displayed similar properties to those of a comigrating, gelatin-cleaving activity present in 69-h-old embryos.

Presence of Cu/Zn superoxide dismutase (SOD) immunoreactivity in neuronal hyaline inclusions in spinal cords from mice carrying a transgene for Gly93Ala mutant human Cu/Zn SOD.

This investigation deals with the immunocytochemical localization of Cu/Zn superoxide dismutase (SOD) in the spinal cord neurons of transgenic mice that overexpress Gly93Ala mutant human Cu/Zn SOD and demonstrate clinicopathological features similar to human amyotrophic lateral sclerosis (ALS) with Cu/Zn SOD mutation. At low magnification of light microscopy, the gray and white matter of the spinal cord of Gly93Ala mice showed more intense Cu/Zn SOD immunoreactivity than that of control mice. At higher magnification, the cytoplasm of control mice neurons displayed a distinct staining for Cu/Zn SOD, whereas the surrounding neuropil was only weakly stained. In contrast, the intensity of Cu/Zn SOD immunoreactivity in the cytoplasm of the majority of Gly93Ala mice neurons was similar to that in the neuropil. Almost all neuronal hyaline inclusions (NHIs) of Gly93Ala mice were positively immunostained by antibodies to Cu/Zn SOD, ubiquitin and phosphorylated neurofilament protein (NFP), the intensities of which were much higher in the NHIs than in the surrounding cytoplasm. In control mice, significant Cu/Zn SOD precipitation was not observed to be limited to any particular region of the neuronal cytoplasm. Intracytoplasmic vacuoles in the neuronal soma and processes of Gly93Ala mice were not stained by any of these antibodies. These results indicate that Cu/Zn SOD colocalizes with ubiquitin and phosphorylated NFP in NHIs of mice expressing mutant Cu/Zn SOD; similar findings have been shown for Lewy body-like inclusions of familial ALS patients with Cu/Zn SOD mutation. Moreover, our results point to the possibility that Cu/Zn SOD mutation may have a role in the abnormal Cu/Zn SOD accumulation in the NHIs, in association with motor neuron degeneration.

Histoenzymologic study of hyalocytes in tissue culture.

The mitochondrias of the hyalocytes contain lactic dehydrogenase but no glucose-6-phosphate dehydrogenase, so that only aerobic respiration is possible. Among the lysosomal enzymes, acid phosphatases and beta-glucuronidase are found, the latter facilitating the turnover of the hyaluronic acid. There is no galactosidase, as the hyaluronic acid of the vitreous does not contain galactose.

Inhibitory activity of alpha 1-antitrypsin in infants with idiopathic respiratory distress syndrome.

In two groups of neonates serum concentrations of alpha-1-antitrypsin (1-AT) were determined by means of radial immunodiffusion, and 1-AT inhibitory capacity determinations were made using benzoyl-1-arginine-p-nitroanilide as a substrate. There were 66 children in the first group of neonates with idiopathic respiratory distress syndrome (IRDS), and 82 healthy children in the second group with birth weight and gestational and post natal ages similar to those of neonates with IRDS. In the latter, 1-AT concentration and inhibitory activity levels were lower than those found in healthy children; these differences were statistically significant. These findings explain both the retardation in the lysis of the hyaline membrane and the fact that a decrease in serum 1-AT levels elicits lung tissue damage by the action of proteolytic enzymes contained in leucocytes.

Enzyme histochemical studies on the formation of hyalin bodies in the epithelium of odontogenic cysts.

Frozen sections of eight odontogenic cysts, including one keratocyst, were incubated to show the following enzyme activities: NADH2 diaphorase, NADPH2 diaphorase, glucose-6-phosphate dehydrogenase, acid phosphatase and leucine aminopeptidase. The disbribution of lipid was shown by the oil red 0 method. The activities of all three oxidative enzymes were strongest in epithelial cells bordering hyalin bodies and in basal cells in the epithelial lining. Hydrolytic enzyme activity was absent from all but the most superficial epithelial cells but was present in macrophages and, in lesser amounts, in granular material in the same sections. The granular material frequently contained lipid. The lack of hydrolytic enzyme activity in bordering epithelial cells is inconsistent with the theory that hyalin bodies form from degenerating blood vessels. High aerobic oxidative enzyme activity in the same cells also conflicts with the concept that the bodies are a keratinous product. The findings lend support to the theory that hyalin bodies are an epithelial secretion.