Purification and various properties of asialoglycoprotein receptors from the mouse liver

The receptor for asialoglycoproteins was isolated from murine liver and was purified by means of biospecific chromatography on sepharose-Asialo-orosomucoid. The obtained receptor with an absorption maximum at 277 nm binds to the nonreducing terminal galactosyl residues of glycoproteins similar to the receptors from liver of other mammalians. The interaction between this receptor and desialylated glycoproteins requires the presence of calcium. The dependence of specific binding on the concentration of [125I]acialo-orosomucoid used as a ligand gives a saturating curve. The dissociation constant for the receptor-ligand complex is 0.4 X 10(-9) M. Similar to asialo-orosomucoid, the receptor binds the p-aminophenyl-beta-D-galactopyranoside derivatives of bovine serum albumin, ovalbumin and acid alpha-glucosidase synthesized by us earlier. Possible use of the asialoglycoprotein receptor as a highly specific carrier transporting the modified acid alpha-glucosidase to hepatocyte lysosomes is discussed.     

Influence of crop rotation and intercropping of cassava with legumes on VA mycorrhizal symbiosis of cassava

Summary In comparison to cassava grown in monoculture the root infection of cassava with vesicular-arbuscular mycorrhiza was increased by crop rotation with grain legumes in the field. This was also found when cassava was intercropped with legumes and fertilized. A possible specificity of mycorrhizal fungi to increase the yield of one species more than the other when grown in association, is discussed.     

An isozyme-specific selective system for the recovery of mammalian cells deficient in hepatic alcohol dehydrogenase activity

A selective system toxic towards mammalian cells expressing the liver-specific isozyme of alcohol dehydrogenase (L-ADH) has been developed. A number of alpha-unsaturated primary and secondary alcohols were assayed for their ability to serve as substrates for rat liver ADH and were screened for cytotoxicity towards L-ADH+ and L-ADH- cells. 1-Propen-3-ol and 1-penten-3-ol were identified as agents showing selective cytotoxicity. Reconstruction experiments demonstrated that 1-propen-3-ol at a concentration of 15 microM could be used to recover L-ADH- clones from mixed populations of L-ADH+ and L-ADH cells. Cells expressing the non-allelic S-ADH isozyme were not killed under these conditions. The selective system defined in this report is thus isozyme-specific.     

Characterization of the microtubule movement produced by sea urchin egg kinesin

We have used an in vitro assay to characterize some of the motile properties of sea urchin egg kinesin. Egg kinesin is purified via 5'-adenylyl imidodiphosphate-induced binding to taxol-assembled microtubules, extraction from the microtubules in ATP, and gel filtration chromatography (Scholey, J. M., Porter, M. E., Grissom, P. M., and McIntosh, J. R. (1985) Nature 318, 483-486). This partially purified kinesin is then adsorbed to a glass coverslip, mixed with microtubules and ATP, and viewed by video-enhanced differential interference contrast microscopy. The microtubule translocating activity of the purified egg kinesin is qualitatively similar to the analogous activity observed in crude extracts of sea urchin eggs and resembles the activity of neuronal kinesin with respect to both the maximal rate (greater than 0.5 micron/s) and the direction of movement. Axonemes glide on a kinesin-coated coverslip toward their minus ends, and kinesin-coated beads translocate toward the plus ends of centrosome microtubules. Sea urchin egg kinesin is inhibited by high concentrations of SH reagents ([N-ethylmaleimide] greater than 3-5 mM), vanadate greater than 50 microM, and [nonhydrolyzable nucleotides] greater than or equal to [MgATP]. The nucleotide requirement of sea urchin egg kinesin is fairly broad (ATP greater than GTP greater than ITP), and the rate of microtubule movement increases in a saturable fashion with the [ATP]. We conclude that the motile activity of egg kinesin is indistinguishable from that of neuronal kinesin. We propose that egg kinesin may be associated with microtubule-based motility in vivo.     

Staining of viable and nonviable myotubes and of myofibrils by the fluorescent dye merocyanine 540

Merocyanine 540 (MC 540) has been reported to interact specifically with excitable plasma membranes in live cells [3]. Here we show that the MC 540 fluorescence staining pattern previously believed to be characteristic of viable myotubes [3] is observed in formaldehyde-fixed cells. In contrast, viable myotubes show an MC 540 fluorescence staining pattern that is characteristic of cell surface staining (no internal structures fluoresce). The specific I-band and H-zone fluorescence of isolated myofibrils is also consistent with the interpretation that the fluorescence patterns previously reported for viable myotubes are in fact characteristic of cells with disrupted plasma membranes. Time-course observations of MC 540 and trypan blue staining of myotubes suggest that when plasma membrane integrity is lost, MC 540 fluorescence can be visualized inside the cell 5-10 min before trypan blue absorbance. Thus the trypan blue viability assay can be misleading when applied to myotubes.     

Modulation of antigen-induced T cell proliferation by alpha 2M-trypsin complexes

Proteinase-complexed alpha 2-macroglobulin (alpha 2M) could be shown to interfere with T cell proliferation in response to antigen presented by autologous antigen-pulsed monocytes (M phi) (antigen-induced M phi-T cell interaction, MTI). Addition of alpha 2M-trypsin (alpha 2M X T) complexes to cultures of T cells and antigen-pulsed M phi led to a dose-dependent decrease of T cell proliferation (up to 91% inhibition of the T cell response), whereas the same concentrations of free (native) alpha 2M had no effect on antigen-induced MTI. The observed interference with MTI could be attributed to residual enzymic activity of the alpha 2M X T complex. Addition of aprotinin, a low Mr protein proteinase inhibitor able to penetrate to the enzyme entrapped within the alpha 2M molecule and thus bind to and inactivate the enzyme's active site, resulted in a reversal of the alpha 2M X T-induced biological effect. Inactivation of the enzyme's active site within alpha 2M X T was monitored by a decrease in the hydrolytic activity of the complex. Kinetic studies (addition of alpha 2M X T 24 to 48 hr after culture onset was shown to be still inhibitory) indicated an effect at the level of the T cell or its mediators, but an overnight incubation of T cells with alpha 2M X T did not alter these cells' capacity to proliferate in response to an antigenic stimulus. An additional effect of alpha 2M X T on the antigen-presenting cell cannot be ruled out at present. However, alpha 2M X T did not alter the percentage of monocytes expressing HLA-DR, -DP, and -DQ or interfere with interleukin 1 release if added to M phi at concentrations that significantly inhibited MTI. Furthermore, incubation of M phi with alpha 2M X T for 1 hr before antigen pulsing had no effect on the M phi antigen presenting capacity.     

Effect of the antioxidant dibunol on the function of the adrenal cortex, the thyroid and the adenohypophysis in adult and old rats

The effect of a single antioxidant (dibunol-D) injection (100 mg/kg body weight) on the functional activity of adrenal cortex (AC), thyroid gland (TG) and tropic hormone production by adenohypophysis (AH) has been studied in old and adult rats. For 48 hours following D administration two-phase changes in adrenocorticotropic function of AH and steroidogenesis were detected in the AC: activation during the first hours was followed by suppression 24 hours later, and recovery 48 hours later. Thyrotropic AH function and thyroidogenesis were found to be decreased during the first hours of D effect. Thyroidogenesis recovery by the end of the first day was delayed as compared to the recovery of AH thyrotropic function. It is suggested that the mechanisms of D action are based on its effects mediated by changes in the functional activity of endocrine glands and associated with resetting of endocrine regulation of body functions.