Endocytosis of aggregated immunoglobulin G by rat basophilic leukemia cells; rate, extent, and effects on the endocytosis of immunoglobulin E

Rat basophilic leukemia (RBL) cells have distinct receptors for IgE and IgG. We assessed the endocytosis of chemically and immunochemically cross-linked mouse-IgG and its influence on the simultaneous endocytosis of IgE. We found that at 37 degrees C, aggregates of IgG and IgE were endocytosed at about the same rate with one-half of the maximal endocytosis occurring in 5 to 13 min, and the efficiency of endocytosis for both ligands ranging from 40 to 70%. We also found that endocytosis of cross-linked IgE and IgG occurred simultaneously and neither ligand significantly affected the rate or extent of endocytosis of the other. The cells accumulated the cross-linked IgG, and then released it to the extracellular environment, at a rate (less than 3%/hr) slower than the released endocytosed IgE (greater than 10%/hr). Using an assay that discriminates between unbound and receptor-bound oligomeric IgG, we found that oligomeric IgG is endocytosed with its receptor, and that the bulk of the ligand remains bound to its receptor for greater than 120 min after endocytosis. The differences in the rate of release of endocytosed IgG vs IgE suggests that the intracellular fate or pathway of these two oligomeric ligands may differ.     

Proline hydroxylation of collagens synthesized at different temperatures in vivo by two poikilothermic species

Extent of prolyl hydroxylation in newly synthesized viper collagen is decreased at 10 degrees C to approximately 23% of normal on skin and to approximately 57% of normal in bone collagen. At 20 degrees C, prolyl hydroxylation is approximately 50% of normal in skin and normal in bone. At 10 degrees C and 20 degrees C, prolyl hydroxylation is decreased approximately 32% in the skin collagen of carp. In contrast, prolyl hydroxylation is unchanged at 10 and 20 degrees C in bone, scale and lepidotrichia. Prolyl hydroxylation of cartilaginous endoskeleton showed an approximately 25% decrease at 20 degrees C.     

Immunochemical subfractionation of a microsomal fraction of rat liver with antibody-coated Staphylococcus aureus cells

The procedure for immunochemical adsorption of vesicles with specific antigen on their outer surfaces was improved. When microsomal vesicles were mixed with Staphylococcus aureus cells coated with the antibody against NADPH-cytochrome c reductase, more than 90% of the enzyme activity was adsorbed on the cell, whereas, only about 10% of the activity was adsorbed on cells coated with the same amount of anti-ovalbumin antibody. NADH-cytochrome c reductase and aldehyde dehydrogenase activities were adsorbed on the cell to the same extent as was NADPH-cytochrome c reductase activity. Under this condition, there was no adsorption of the activities of the marker enzymes of lysosomes and Golgi apparatus, whereas large amounts of the activities of the plasma membrane enzymes were adsorbed. The specific activity of NADPH-cytochrome c reductase in the adsorbed vesicles from the microsomal fractions increased considerably. In contrast, marker enzymes of the Golgi or of the plasma membranes could be enriched in unadsorbed vesicles from the Golgi fractions.     

Acid proteinase activity in fish--I. Comparative study of extraction of cathepsins B and D from Mujil auratus

Acid catheptic activity was measured in crude extracts of muscle, liver, heart, spleen and gonads from the fishes Mujil auratus, Sparus aurata and Lightonatus mormyrus. The spleen was the organ which showed the highest activity. A comparative study of the seven most commonly used extraction methods was made. Some were modified to account for the characteristics of the fish organs and the activity extracted from them. The Siebert method resulted as the best extraction method only if 1 mM EDTA was present in the medium. The activity from Mujil auratus muscle was strongly inhibited by iodoacetate, N-ethylmaleimide, p-hydroxy mercuribenzoate, and diazo-acetyl-DL-norleucine methyl ester. The results indicated the presence of a carboxyl-proteinase and a thiol-proteinase. According to inhibition studies, the levels of proteinase and amidase activities shown by different organs of Mujil auratus were re-examined. The spleen extract showed the maximum activity for both cathepsins, but muscle extract accounted for more than 95% of total catheptic activity.     

Spectrophotometric assay of phenolpthalein in biological fluids.

An assay for phenolphthalein in biological fluids has been developed utilizing methods previously applied to the assay of bromosulphalein and to the deconjugation of steroidal compounds in urine. Intestinal perfusate, serum, and urine samples containing phenolphthalein are deproteinized with acidified acetone, the samples dried, and the phenolphthalein redissolved in ethanol. Color is developed with 0.5 m glycine buffer, pH 12, and the samples read at 550 nm after blanking the spectrophotometer with one of the replicates to which acidic glycine buffer is added. To measure conjugated phenolphthalein in urine, the sample is incubated overnight with β-glucuronidase/arylsulphatase prior to phenolphthalein determination as noted above. This method gives an accurate assay of phenolphthalein to 10^?5m concentrations with coefficients of variation between 2 and 8% and with no resulting interference from hemoglobin or bilirubin.     

Segregation Distortion of Marker Nuclear Genes in Alloplasmic and Isoplasmic Lines of Barley

Inheritance of barley nuclear genes responsible for various morphological marker traits was studied in hybrid populations F₂ and F_a. Nine marker genes showed deviation from Mendelian monogenic inheritance depending on the cross direction and maternal cytoplasm. Segregation biases to both recessive mutant and dominant normal phenotypes were observed. Mechanisms of the segregation bias related to cytoplasm substitution in iso- and alloplasmic lines are discussed.     

Induction of erythroid differentiation by cytoplast fusion in mouse erythroleukemia (Friend) cells

An intracellular activity, which is induced by dimethyl sulfoxide (DMSO) or hexamethylenebisacetamide (HMBA) and leads to erythroid differentiation in mouse Friend cells, was characterized by cell fusion between genetically marked intact cells and cytoplasts. For this, a procedure for rapid selection of cybrids was devised by sensitizing non-fused cells with oligomycin. We were able to demonstrate that cytoplasts derived from DMSO- (or HMBA)-treated cells trigger erythroid differentiation upon fusion with UV-irradiated cells. The activity in the cytoplasts remained only transiently and its induction was inhibited by biologically active phorbol esters or cycloheximide. The activity, however, was not induced in cytoplasts by directly treating them with DMSO (or HMBA). These results indicate that (1) the intracellular erythroid-inducing activity is located in cytoplasts, (2) it acts in trans and induces erythroid differentiation as a dominant factor and (3) its production requires de novo nuclear protein synthesis. The mechanisms of the induction of the intracellular activity and of how it triggers erythroid differentiation are discussed.