Synthesis and maturation of cross-reactive glycoprotein in fibroblasts deficient in arylsulfatase a activity

The biosynthesis of arylsulfatase A was studied in cultured fibroblasts by pulse-chase labeling with [2-³H]mannose; the enzyme was isolated by immunoprecipitation and denaturing polyacrylamide gel electrophoresis. In normal fibroblasts, and in fibroblasts from a patient with multiple sulfatase deficiency, the enzyme was synthesized as a glycoprotein of apparent molecular weight of 59,000; half of it was processed over a period of 4 days to M_r= 57,000. The precursor chain of M_r= 59,000 was secreted in the presence of 10 mM NH₄Cl. An immunoprecipitable glycoprotein of normal size was synthesized by fibroblasts from two unrelated patients with metachromatic leukodystrophy, but this material disappeared within twenty hours. In fibroblasts from an individual with pseudodeficiency of arylsulfatase A, the immunoprecipitable precursor glycoprotein was smaller (M_r= 56,000). The synthesis of cross-reactive proteins with altered properties supports the concept of allelic mutations as the genetic basis of metachromatic leukodystrophy and of arylsulfatase A pseudodeficiency.     

The use of amphotericin B to detect inhibitors of cellular cholesterol biosynthesis

Pores formed in the membranes of animal cells by complexes of sterols and the polyene antibiotic amphotericin B can efficiently kill the cells. Thus, in the absence of exogenous sources of cholesterol, inhibitors of enzymes in the cholesterol biosynthetic pathway render cells resistant to amphotericin B. Preincubation of Chinese hamster ovary cells with compactin or 25-hydroxycholesterol, inhibitors of the synthesis of the key intermediate mevalonate, protected cells from amphotericin B killing and this protection was reversed by the addition of exogenous mevalonate. The ability of compactin to confer amphotericin B resistance on normal cells was abolished when cells were provided exogenous cholesterol by the receptor-mediated endocytosis of low density lipoprotein. Low density lipoprotein receptor-defective Chinese hamster ovary cells were not subject to this low density lipoprotein-dependent amphotericin B killing. Exogenous mevalonate did not prevent 4,4,10 beta-trimethyl-trans-decal-3 beta-ol, an inhibitor of mevalonate conversion to sterols, from protecting cells from amphotericin B. A simple two-step protocol in which cells are preincubated (15-24 h) with potential inhibitors and then treated (3-6 h) with amphotericin B was devised to provide a sensitive method for detecting direct (e.g., competitive) and regulatory inhibitors of cholesterol biosynthesis. This protocol may prove useful in detecting potential antihypercholesterolemia drugs and is currently being used to isolate mutants in receptor-mediated endocytosis.     

Detection of beta-adrenergic receptors on rabbit mononuclear cells isolated free of significant contamination by other cell types

In order to study rabbit mononuclear cell surface receptors, it was necessary to develop a procedure to isolate mononuclear cell preparations that are free of significant contamination by other cell types, especially platelets. Centrifugation of dextran-sedimented, anti-coagulated whole blood through Hypaque (density 1.060) at 600 X g for 5 min at 22 degrees C eliminated greater than 93% of starting platelets. A second 5-min Hypaque centrifugation of Hypaque-Ficoll-isolated mononuclear cells (MNC) (approximately 80% lymphocytes) at 450 X g for 5 min at 22 degrees C reduced platelet contamination to less than one platelet per three MNC, and resulted in the overall removal of greater than 99.5% of starting platelets. These relatively pure MNC which were isolated in less than 2 hr were identified as having beta-adrenergic receptors by radioligand binding techniques using [125I]iodohydroxybenzylpindolol [( 125I]IHYP). Binding of [125I]IHYP to intact rabbit MNC was a saturable, stereospecific, and rapid process with a dissociation constant (KD) of 0.53 +/- 0.18 nM and a binding capacity of 3,461 +/- 235 sites/cell.     

A novel opioid peptide, leumorphin, acts as an agonist at the kappa opiate receptor

The primary structure of the common precursor of porcine beta-neo-endorphin and dynorphin (preproenkephalin B) has shown the existence of a third leucine-enkephalin (leu-enkephalin) sequence with a C-terminal extension of 24 amino acids. This nonacosapeptide, named leumorphin, was approximately 70 times more potent than leu-enkephalin in inhibiting the contraction of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum. This action of leumorphin, like those of beta-neo-endorphin and dynorphin, was antagonized less effectively by naloxone than that of leu-enkephalin, but more effectively by Mr2266, an antagonist relatively specific for the kappa type opiate receptor. The inhibitory action of leumorphin or beta-neo-endorphin on the contraction of the guinea pig ileum muscle strip was reduced in a dose-dependent manner by pretreatment with dynorphin and vice versa. Leumorphin as well as beta-neo-endorphin and dynorphin inhibits the contraction of the rabbit vas deferens which is known to have only the kappa type opiate receptor. This action was also effectively antagonized by Mr2266. It is concluded that leumorphin has potent opioid activity and acts at the kappa receptor, like other opioid peptides derived from preproenkephalin B.     

Somatostatin mydriasis in mice

Administration of somatostatin intracerebroventricularly to mice produced a dose-dependent mydriasis, whereas intravenous injections were ineffective. Naloxone could prevent or abolish this effect. It is suggested that somatostatin either directly excites opiate receptors or activates endopioid pathways involved in the regulation of pupillary size.     

Root development and configuration in intensively managed radiata pine plantations

Summary In south-east Australia, where radiata pine (Pinus radiata D. Don) is grown on sandy soils low in nutrients and short of water, early establishment, and rapid growth to canopy closure lead to increased productivity. At this stage demands for nutrients and water are high, and trees respond vigorously to silvicultural inputs.For several months after transplanting in winter roots are confined within a narrow planting wedge, low temperature restricts new root growth and slows recovery from water stress in plants. From spring, depending upon the configuration and vigour of the roots transplanted, lateral roots extend radially throughout the soil.Although there were small decreases in concentration of roots radially from the stems of very young trees, such spatial differences disappeared between ages 2 and 3, so that rooting density was independent of distance from the stem. The pattern of vertical distribution of lateral roots was not influenced by age and 80–90% of the lateral roots were within the top 30 cm soil. Roots developed rapidly as the trees grew towards canopy closure, but in general the rooting densities of these pines are among the lowest reported for plants. In rapidly growing trees approaching canopy closure, the secondary thickening of the lateral roots was sufficient to double the weight of roots without altering root length.Knowledge about root growth and root configuration during the early phase of plantation development will assist management decisions where intensive silviculture is practiced, and hence ensure the most efficient use of nutrients and water.     

Effect of Na+, K+, ouabain,amiloride and ethacrynic acid on the transepithelial potential across Malpighian tubules of Locusta

A transepithelial potential of +8.74±0.29 mV (n = 85) has been recorded across the Malpighian tubules of Locusta. The effect of varying the Na⁺ and K⁺ concentration in the bathing medium on the transepithelial potential has been determined. The data show that the transepithelial potential does not obey the Nernst equation for K⁺. Ouabain, ethacrynic acid and amiloride all inhibit the transepithelial potential. The results are discussed in relation to the nature of the mechanisms of cation transport across the Malpighian tubules.     

Inhibition of the graft-versus-host response by BCGcw-induced suppressor cells or prostaglandin E1

Immunization of C57BL/6 mice with BCGcw stimulated a population of "suppressor cells" which had a decreased capacity to induce the graft-versus-host response. The graft-versus-host response was quantitated using the Simonsen splenomegaly assay. F1 mice (C57BL/6 X CBA) were inoculated intraperitoneally with 1 X 10(8) parental (C57BL/6) or (CBA) spleen cells. The F1 mice were sacrificed 13 days later and the resulting splenomegaly was 3-4 times the normal amount. F1 mice which were injected with parental BCGcw-primed C57BL/6 spleen cells had a 50% inhibition of splenomegaly, whereas BCGcw-primed CBA spleen cells (a strain which does not develop suppressor cells) did not show this inhibition. In vitro results also confirmed that only C57BL/6 mice and not CBA mice developed suppressor cells after BCGcw immunization. A second study showed that X-irradiated (1000 R) BCGcw-primed "suppressor cells" could inhibit splenomegaly caused by the inoculation of normal parental C57BL/6 cells into F1 mice. The mechanism by which BCGcw-primed "suppressor cells" caused this inhibition of splenomegaly was delineated and found to be dependent upon the secretion of prostaglandin (PGE-1). Indomethacin and aspirin, potent inhibitors of prostaglandin synthesis, blocked the activity of C57BL/6 BCGcw "suppressor cells" and splenomegaly resulted. Systemic administration of the prostaglandin (15S)-15-methyl PGE-1 reduced splenomegaly approximately 50% in F1 mice which were injected with C57BL/6 or CBA cells. These results indicated that immunization with BCGcw stimulated a population of "suppressor cells" which could cause a decrease in graft-versus-host response and that the secretion of prostaglandin was responsible for this inhibition.     

Lack of orthogonal particle assemblies and presence of tight junctions in astrocytes of the goldfish (Carassius auratus)

Summary Specific antibodies raised against a human 28 000 dalton cerebellar calcium-binding protein (CaBP) were used in an immunocytochemical study during development of the rat cerebellum. Both light and electron microscopy showed (1) that labelling was entirely restricted to the Purkinje cells, (2) that it appeared very early in Purkinje cell development, (3) that the entire cell was labelled from the tip of the smallest dendrites to the axonal terminals, and (4) that with increasing age, the immunoreaction appeared to be progressively restricted to the cell and organelle membranes.