Oxidative inactivation of the calcium-stimulated neutral proteinase from human red blood cells by divicine and intracellular protection by reduced glutathione

Calpain, the micromolar Ca2+-requiring form of Ca2+-stimulated neutral proteinase purified from human red cells, is remarkably inactivated during autoxidation of divicine (2,6-diamino-4,5-dihydroxypyrimidine), an aglycone implicated in the pathogenesis of favism. Inactivation of purified calpain is produced, in decreasing order of efficiency, by transient, probably semiquinonic species arising from autoxidation of divicine, by the H2O2 that is formed upon autoxidation itself, and by quinonic divicine, respectively. Purified procalpain, the millimolar Ca2+-requiring form that can be converted to the fully active calpain form by a variety of mechanisms, is less susceptible than calpain itself to inactivation by the same by-products of divicine autoxidation. When intact red cells are exposed to autoxidizing divicine, procalpain undergoes a significant loss of activity. At 1 mM divicine, intracellular inactivation is observed with procalpain only, while the activity of a number of red cell enzymes is unaffected. Inactivation of procalpain is consistently greater in red cells from glucose-6-phosphate dehydrogenase-deficient subjects than in normal cells. Restoration of normal levels of glucose-6-phosphate dehydrogenase activity by means of entrapment of homogeneous human glucose-6-phosphate dehydrogenase in the deficient red cells results in normal stability of intracellular reduced glutathione; decreased susceptibility of procalpain to inactivation by autoxidizing divicine. These findings suggest that in the glucose-6-phosphate dehydrogenase-deficient red cells the procalpain-calpain system is a major target of divicine cytotoxicity.     

Identification of carrageenan in mammalian tissues: An analytical and histochemical study

Synopsis Methods have been developed for the analytical estimation and histochemical demonstration of carrageeman in the granuloma induced in rats and guinea-pigs by subcutaneous injection of degraded carrageenan.The analytical method for the determination of carrageenan in tissues involved a preliminary clean-up procedure. The tissues were defatted by solvent extraction and incubated with papain and trypsin to remove proteins. Carrageenan and naturally occurring acid mucopolysaccharides were isolated using cetyl pyridinium chloride. The subsequent separation and estimation of carrageenan was carried out by electrophoresis on cellulose acetate paper. Following electrophoresis the cellulose acetate strips were incubated with hyaluronidase to remove acid mucopolysaccharides, and stained with Toluidine Blue. The stained band corresponding to pure degraded carrageenan was quantitated on a scanning densitometer. The method was capable of detecting 0.25 g of degraded carrageenan in tissue.The most suitable method for the histochemical demonstration of carrageenan in paraffin embedded tissues was found to be Alcian Blue at either pH 1 or a CEC (critical electrolyte concentration) of 1.0 M MgCl₂ (pH 5.8). At this pH or CEC, both the carrageenan and the strongly acidic glycosaminoglycans were stained. Prior digestion with hyaluronidase and neuraminidase eliminated the staining of the tissue polysaccharides so that the carrageenan could be visualized within macrophages and in the extracellular space. Mast cell granules retained their staining properties after mucolytic digestion; but morphologically, mast cells could be distinguished from macrophages containing carrageenan.     

A RADIOIMMUNOASSAY FOR MEASURING 14-3-2 PROTEIN IN CELL EXTRACTS

—A rapid and sensitive radioimmunoassay for the neuron-specific protein, 14-3-2, has been developed. The assay was used to determine the amounts of 14-3-2 protein in soluble extracts prepared from clones of the C 1300 murine neuroblastoma and from C 1300 × L cell hybrid clones. The values obtained with the radioimmune procedure correlated with those obtained in microcomplement fixation and microprecipitin assays.     

THE RELATIONSHIP BETWEEN RNA SYNTHESIS AND HEMOGLOBIN SYNTHESIS IN AMPHIBIAN ERYTHROPOIESIS : Cytochemical Evidence

A cytochemical study of the relationship between RNA synthesis and hemoglobin synthesis has been performed on splenectomized newts, Triturus viridescens. Employing radioautography, labeled cytidine was incorporated into the RNA of the early developmental stages but was not incorporated in the later stages. Labeled leucine was incorporated into the cellular protein of all stages except mature erythrocytes but was incorporated at a higher level in the later stages. Microphotometric measurements of azure B binding to cytoplasmic RNA revealed a sharp initial increase between the stem cell and proerythroblast followed by a rapid decrease between the basophilic and polychromatophilic stages. The loss of cytoplasmic RNA became more gradual in the late stages and, in the mature erythrocyte, little or no cytoplasmic RNA could be detected. Measurements of cytoplasmic total protein, using fast green staining at pH 2.0, and of heme showed that both curves increased similarly with development, indicating net hemoglobin synthesis. The results are compatible with the hypothesis that, as the stem cell differentiates along erythrocytic lines, a stable "messenger" RNA specifying the production of a given type or types of hemoglobin is formed. This complex probably becomes associated with ribosomal RNA and is retained throughout the process of RBC differentiation.     

Elevated Amounts of Protein in C6 Glial Cells During the Growth-Inhibitory Response Elicited by Dexamethasone

Abstract: Dexamethasone suppresses C6 glial cell proliferation in vitro. This growth-inhibitory response is accompanied by elevated amounts of acid-insoluble protein in the steroid-treated cells relative to controls. These results provide additional evidence that the glucocorticoid acts to arrest C6 cell proliferation in G2.     

Homing Behaviour in Polyergus rufescens Latr. (Hymenoptera,Formicidae)

Homing mechanisms of the European slave-making ant Polyergus rufescens Latr. are investigated by field experiments. The analysis of the behaviour and paths of both homing scouts and raiders after passive displacement showed that: i. Scouts probably home by using a path integration system based on celestial cues; and ii. Displaced raiders do not seem to adopt such a vectorial orientation mechanism. Moreover, we found that passively displaced scouts exhibit a systematic search strategy for the nest after a rectilinear path. By contrast, raiders perform a similar search pattern just after release. Similarities between Cataglyphis and Polyergus homing behaviour are discussed.     

Decreased superovulatory responses in heifers superovulated in the presence of a dominant follicle

Dairy heifers were superovulated in the presence (dominant group, N = 8) or absence (non-dominant group, N = 6) of a dominant follicle at the start of a a superovulatory treatment on Days 7-12 of the oestrous cycle (Day 0 = oestrus). Daily ultrasonographic observations of ovaries (recorded on videotape) starting on Day 3 were used to assess the presence or absence of a dominant follicle (diameter greater than 9 mm, in a growing phase or at a stable diameter for less than 4 days) and to monitor follicular development before and during treatment. The number of CL estimated by ultrasonography (7.1 +/- 1.8 vs 13.5 +/- 1.4) or by rectal palpation (6.9 +/- 2.0 vs 16.3 +/- 1.6) and mean progesterone concentrations (32.5 +/- 19 vs 80.7 +/- 16 ng/ml) after treatment were lower (P less than 0.01) in the dominant than in the non-dominant group. Based on number of CL, two populations of heifers were identified in the dominant group, i.e. those that had a high (dominant-high, N = 4; greater than 7 CL) or a low (dominant-low, N = 4; less than 7 CL) response to treatment. During treatment, the increases in number of follicles 7-10 mm and greater than 10 mm in diameter occurred sooner and were of higher magnitude in the non-dominant than in the dominant-high or dominant-low groups (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)