Oxygen free radicals induced release of lysosomal enzymes in vitro

Summary The effect of oxygen free radicals, generated by xanthine and xanthine oxidase, was studied on the release of lysosomal hydrolase from rat liver lysosomes in vitro. A lysosomal enriched subcellular fraction was prepared, using differential centrifugation technique, from the homogenate of rat liver. The biochemical purity of the lysosomal fraction was established by using the markers of different cellular organelles. Oxygen free radicals were generated in vitro by the addition of xanthine and xanthine oxidase. The release of lysosomal hydrolase (-glucuronidase) from the lysosomal fraction was measured. There was a 3 to 4 fold increase in the release of -glucuronidase activity in the presence of xanthine and xanthine oxidase when compared to that in the absence of xanthine and xanthine oxidase. In the presence of superoxide dismutase (SOD), a scavenger of oxygen free radicals, the xanthine and xanthine oxidase system was unable to induce the release of -glucuronidase activity from the lysosomes. Sonication (2 bursts for 15 sec each) and Lubrol (2 mg/10 mg lysosomal protein) treatment, which are known to cause membrane disruption, also induced the release of -glucuronidase from lysosomal fraction. This release of -glucuronidase by sonication and lubrol treatment was not prevented by SOD. These data indicate that lysosomal disruption is a consequence of oxygen free radicals, generated by xanthine and xanthine oxidase.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EGTA Ethylene Glycol Bis-(-aminoethyl ether)N,N,-N,N-tetracetic acid - Tris Tris (hydroxymethyl) aminomethane - SOD Superoxide Dismutase     

Role of the N-terminal region of the A chain in beta 1-bungarotoxin from the venom of Bungarus multicinctus (Taiwan-banded krait)

The N-terminal -amino groups of ₁-bungarotoxin (₁-Bgt) fromBungarus multicinctus venom were modified with trinitrobenzene sulfonic acid and the modified derivative was separated by high performance liquid chromatography. The trinitrophenylated (TNP) derivative contained two TNP groups at the -amino groups of A chain and B chain and showed a marked decrease in enzymatic activity. Methionine residues at positions 6 and 8 of the A chain were oxidized with chloramine T or cleaved with cyanogen bromide to remove the N-terminal octapeptide. Oxidation of methionine residues and removal of the N-terminal octapeptide caused a precipitous decrease in enzymatic activity, whereas antigenicity remained unchanged. The presence of dihexanoyllecithin influenced the interaction between ₁-Bgt and 8-antilinonaphthalene sulfonate (ANS) and revealed that ₁-Bgt consists of two types of ANS-binding sites, one at the substrate binding site of the A chain and the other might be at the B chain. The modified derivatives still retained their affinity for Ca²⁺ and ANS, indicating that the N-terminal region is not involved in Ca²⁺ and substrate binding. A fluorescence study revealed that the -amino group of the A chain was in the vicinity of substrate binding site and that the TNP -amino groups were in proximity to Trp-19 of the A chain. In addition, the study showed that the N-terminal region is important for stabilizing the architectural environment of Trp-19. The results, together with the proposal that Trp-19 of the A chain is involved in substrate binding, suggest that the N-terminal region of the A chain plays a crucial role in maintaining a functional active site for ₁-Bgt.     

Prediction of the packing arrangement of strands in β-sheets of globular proteins

A method is proposed for predicting the adjacency order in which strands pack in a -sheet in a protein, on the basis of its amino acid sequence alone. The method is based on the construction of a predicted contact map for the protein, in which the probability that various residue pairs are close to each other is computed from statistically determined average distances of residue pairs in globular proteins of known structure. Compact regions, i.e., portions of the sequence with many interresidue contacts, are determined on the map by using an objective search procedure. The proximity of strands in a -sheet is predicted from the density of contacts in compact regions associated with each pair of strands. The most probable -sheet structures are those with the highest density of contacts. The method has been tested by computing the probable strand arrangements in a five-strand -sheet in five proteins or protein domains, containing 62–138 residues. Of the theoretically possible 60 strand arrangements, the method selects two to eight arrangements as most probable; i.e., it leads to a large reduction in the number of possibilities. The native strand arrangement is among those predicted for three of the five proteins. For the other two, it would be included in the prediction by a slight relaxation of the cutoff criteria used to analyze the density of contacts.     

The regions of alpha-neurotoxin binding on the extracellular part of the alpha-subunit of human acetylcholine receptor

A set of 18 synthetic uniform overlapping peptides spanning the entire extracellular part (residues 1–210) of the -subunit of human acetylcholine receptor were studied for their binding activity of¹²⁵I-labeled -bungarotoxin and cobratoxin. A major toxin-binding region was found to reside within peptide 122–138. In addition, low-binding activities were obtained with peptides 34–49 and 194–210. It is concluded that the region within residues 122–138 constitutes a universal major toxin-binding region for acetylcholine receptor of various species.     

A reevaluation of the amino acid sequence of human follitropin beta-subunit

A collaborative study from two laboratories has been undertaken to re-evaluate the human follitropin -subunit sequence (hFSH), since areas of uncertainty remain in the wake of two earlier reports. The first report was by Shome and Parlow (1974). The second, by Saxena and Rathnam (1976), proposed revisions for sequence not definitively placed in the first study, as well as some differences in other placements. We have re-examined the sequence of the hFSH with more recent methodology. This has led to revision of certain areas of the sequence and resolution of differences between the two earlier proposals. Specifically, an-Ile-Ser- is established at 21–22, Asp at 41, Arg at 44, Lys at 46, and Glu at 111. These were areas of disagreement in the earlier proposals. A definitive placement of the residues around tryptophan-27 has now been obtained by three laboratories. C-terminal heterogeneity was observed with subunits ending at residue 107, 109, or 111. N-terminal heterogeneity has been observed in all preparations examined to date. A significant population of molecules with a proteolytic nick between residues 38–39 is noted. This is very likely an artifact of the collection and processing. The preparations examined in the present studies showed no evidence of residues 112–118 proposed by Saxena and Rathnam.     

Paracoccidioidomycosis in nude mice: Presence of filamentous forms of the fungus

Congenitally athymic nude mice (nu/nu) and their phenotypically normal littermates (nu/+) were intraperitoneally infected with yeast cells of a strain of Paracoccidioides brasiliensis. The nude mice developed a severe and generalized infection with an intense parasitism of several organs, accompanied by a low-grade of tissue reaction. The lesions were characterized by abundant yeast-like cells of the fungus, and in some animals, numerous hyphal forms could be well visualized. In control animals, infection was moderate, almost exclusively restricted to the area of inoculation, and the lesions presented few parasites surrounded by an inflammatory response. Filamentous forms of the fungus were never encountered in these animals.     

Serotonin and gastrin/cholecystokinin-like immunorcactive neurons in the larval retrocerebral complex of the blowfly Calliphora erythrocephala

Summary The presence and distribution of neurons immunoreactive against antibodies to serotonin (5-HT) and gastrin/cholecystokinin (gastrin/CCK) has been studied in the larval retrocerebral complex of the blowfly Calliphora erythrocephala, a composite structure which consists of the corpus cardiacum, the corpus allatum, the thoracic gland and a portion of the cephalic aorta. Immunoreactive material was found in all these elements except in the corpus allatum. Six to eight cell bodies in the corpus cardiacum and four to eight cell bodies in the thoracic gland were 5-HT immunoreactive (5-HTi). These 5-HTi cell bodies send processes to the neuropil of the corpus cardiacum and to neurohemal sites in the cephalic aorta, corpus cardiacum and ventral part of the thoracic gland. Six to eight cell bodies in the corpus cardiacum and four to six cell bodies in the thoracic gland reacted with antibodies against gastrin/CCK. These cell bodies send processes to the neuropil of the corpus cardiacum and to neurohemal sites in the corpus cardiacum and the cephalic aorta in a pattern resembling that of the 5-HTi fibers. Additional gastrin/CCK-like immunoreactive fibers were shown to come from the central nervous system via the two nervi corporis cardiaci. An electron-microscopical analysis was performed to analyze further the morphological features revealed by the light-microscopic immunocytochemical technique. This confirmed the existence of neurosecretory-like terminals among the gland cells of the thoracic glands and the existence of neurohemal sites in several regions of the larval retrocerebral complex. Some functional aspects of the retrocerebral complex are discussed on the basis of the presented data.     

Fine-structural and immunohistochemical study of anterior pituitary cells of Snell dwarf mice

Summary Snell dwarf mice display remarkable retardation of growth after birth and are known to lack prolactin (PRL), thyroid stimulating hormone (TSH) and growth hormone (GH). The aim of this study was to determine the reason for these hormonal deficiencies. We examined the fine structure of the gland and its immunohistochemical staining pattern with respect to antisera raised against PRL, TSH, GH, adrenocorticotrophic hormone (ACTH) and luteinizing hormone (LH). The gland of control mice reacted immunohistochemically against all antisera used, whereas only ACTH-producing cells (ACTH cells) and LH-producing cells (LH cells) were distinguished in the dwarf mice. ACTH cells in dwarf mice varied in cell shape, although they were similar in size to those of controls. The distribution of secretory granules in the cytoplasm varied from cell to cell. LH cells in the dwarf mice showed immature features, having poorly developed rough endoplasmic reticulum and Golgi apparatus. The cells were about half the size of controls, and secretory granules were smaller. In dwarf mice, non-granulated cells were encountered in addition to granulated ACTH and LH cells. Some of them formed small clusters, characteristic cell junctions being found between the cells; they thus appeared to be follicular cells. The above results suggest that hormone deficiency in Snell dwarf mice is a result of a defect in the hormoneproducing cells in the gland.     

Atropine inhibits the degranulation of Paneth cells in ex-germ-free mice

Summary Previous studies have shown that the secretory products of Paneth cells contain antibacterial agents (lysozyme, IgA) that are affected by the bacterial milieu in the intestine. To investigate whether Paneth-cell secretion is controlled via cholinergic mechanisms, the ultrastructure of Paneth cells was studied in four animal groups: (1) germfree (GF) control mice (Jcl: ICR [GN], male, 13 weeks old), (2) GF mice injected subcutaneously with atropine sulfate (200 mg/kg body weight, dissolved in physiological saline 20 mg/ml), (3) ex-GF mice inoculated with feces from specific-pathogen-free (SPF) mice, and (4) ex-GF mice injected with atropine and inoculated with feces from SPF mice. In ex-GF mice inoculated with feces, 70–90% of the Paneth cells showed fewer secretory granules than those from GF mice (p<0.01). Approximately 30% of the Paneth cells had a large vacuole (3–10 m diameter) in the apical cytoplasm. Exocytosed electron-dense material from secretory granules was observed in a few crypt lumens. In ex-GF mice inoculated with feces and given atropine, about 90% of the Paneth cells contained numerous secretory granules, like those in GF control mice, but vacuolated Paneth cells and exocytotic figures were rare; thus the secretion of Paneth cells was blocked by atropine. It is therefore possible that the bacterial milieu in the intestine affects the secretory activity of Paneth cells via cholinergic mechanisms.