Immunogenicity of subcellular fractions and molecular species of MuLV-induced tumors

Summary YBA, a Moloney virus-induced leukemia in CBA mice, and a relatively weak immunogenic tumor, was screened for the presence of immunogenic antigens. The tumor was subjected to homogenization and subcellular fractionation on sucrose gradients; the immunogenic subcellular fractions underwent further separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The immunogenicity of the subcellular fractions and the SDS-PAGE-isolated molecular species were tested by (their) subcutaneous injection into syngeneic mice and examination of their splenocytes examined against tumor cell and normal cell targets by the chromium release cell-mediated lympholysis assay. Tumor cell homogenates were also separated by SDS-PAGE and tested for immunogenicity without prior fractionation.Splenocytes from mice that had received injections of certain SDS-PAGE-isolated epitopes derived from YBA tumor homogenate or its light and heavy subcellular fractions generated effective cytotoxic responses against YBA target cells after 6 days in vitro cultivation. In contrast, intact YBA tumor cells or non-separated tumor homogenates failed to induce an efficient cytotoxic response. The effector cells induced with the immunogenic SDS-PAGE-isolated epitopes of YBA tumor were specific, since they cytolysed the homologous target cells more efficiently than unrelated target cells or syngeneic normal cells. The activity of these effector cells was affected by varying the effector : target ratio. Augmentation of the cytotoxic responses was obtained when the splenocytes of mice immunized with SDS-PAGE-isolated epitopes of YBA tumor were restimulated in vitro, with the homologous neoplastic cells.Immunogenic SDS-PAGE epitopes were isolated from YAC tumor also (YAC is a Moloney-induced tumor of A mice). The effector cells induced with these separated epitopes were characterized as thymus-derived cells and not as natural killer cells.The results suggest that (1) the molecular repertoire of YBA and YBA tumors contain immunogens that can induce a specific antitumor cell-mediated response; (2) the isolated molecular species injected are more efficient immunogens than the entire, unseparated homogenate sample or a dose of 10⁸ intact inactivated tumor cells; and (3) the gel matrix may be responsible for the enhanced cell-mediated response induced against the weakly immunogenic tumor.     

The distribution of molecular species of phosphatidylinositol in ox brain and its subcellular fractions

1. The phosphatidylinositol content of white and grey matter of ox cerebral hemispheres did not differ. The phosphatidylinositol from grey matter was slightly enriched in palmitic acid and arachidonic acid, and that from white matter was enriched in eicosatrienoic (C(20:3)) acid. These regional differences were apparently due to the greater content of myelin in the white matter, since the same tendencies were observed when combined myelinic and non-myelinic subcellular fractions prepared from the cerebral hemispheres were compared. 2. Purified phosphatidylinositol was converted into its triacetylated methylated derivative and resolved to its molecular species by t.l.c. on AgNO(3)-impregnated silica gel. The tetraenoic molecular species was predominant in phosphatidylinositol from ox cerebral hemispheres, and this feature characterized all the phosphatidylinositol samples extracted from its regions or subcellular fractions. The grey matter was more enriched in the tetraenoic species and the white matter in the trienoic species. 3. The molecular-species composition of phosphatidylinositol from the subcellular fractions of ox cerebral hemispheres was studied. The trienoic species constituted nearly one-fifth of the phosphatidylinositol from two myelinic fractions. ;Large myelin' was more enriched in this species than was ;small myelin'. Both fractions also contained greater concentrations of the dienoic species than the non-myelinic subcellular fractions. The latter fractions, one containing nuclei and the other nerve endings plus mitochondria, were enriched in the monoenoic and tetraenoic species of phosphatidylinositol. The post-mitochondrial supernatant exhibited a pattern of distribution of phosphatidylinositol species intermediate between the myelinic and non-myelinic fractions.     

Translation and identification of the viral mRNA species isolated from subcellular fractions of vesicular stomatitis virus-infected cells.

The cytoplasm of vesicular stomatitis virus (VSV)-infected BHK cells has been separated into a fraction containing the membrane-bound polysomes and the remaining supernatant fraction. Total poly(A)-containing RNA was isolated from each fraction and purified. A 17S class of VSV mRNA was found associated almost exclusively with the membrane-bound polysomes, whereas 14,5S and 12S RNAs were found mostly in the postmembrane cytoplasmic supernatant. Poly(A)-containing VSV RNA synthesized in vitro by purified virus was resolved into the same size classes. The individual RNA fractions isolated from VSV-infected cells or synthesized in vitro were translated in cell-free extracts of wheat germ, and their polypeptide products were compared by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The corresponding in vivo and in vitro RNA fractions qualitatively direct the synthesis of the same viral polypeptides and therefore appear to contain the same mRNA species. By tryptic peptide analysis of their translation products, the in vivo VSV mRNA species have been identified. The 17S RNA, which is compartmentalized on membrane-bound polysomes, codes for a protein of molecular weight 63,000 (P-63) which is most probably a nonglycosylated form of the viral glycoprotein, G. Of the viral RNA species present in the remaining cytoplasmic supernatant, the 14.5S RNA codes almost exclusively for the N protein, whereas the 12S RNA codes predominantly for both the NS and M proteins of the virion.     

Lysolecithinase activity in subcellular fractions of rat organs.

Lysolecithinase activity was measured in subcellular fractions of rat liver, kidney, lung and intestine and compared to similar findings in brain. To obtain optimal assay conditions, each fraction was subjected to a kinetic analysis in the absence and presence of albumin. Among the particulate preparations, lysolecithinase activity of the intestine exceeded by far similar fractions of other organs. Among the soluble fractions, the 100,000xg supernatant of lung had the highest activity. Under the assay conditions used, most of the lysolecithinase activity of all organs was particulate.     

Phospholipase activities in subcellular fractions of human platelets.

A homogenate of human platelets was fractionated by zonal ultracentrifugation into membranes, various granules and mitochondria. The membrane fraction was composed of two populations. The first, which represented 75% of the proteins, was rich in plasma membranes; the second, which represented the remaining 25%, was rich in microsomal membranes. Lysophospholipase was essentially localised in the cytosol. Phospholipase A1 which was only weakly bound to membranes, was mostly found in the soluble fraction (75%); the remainder was located in the plasma membranes and the mitochondria. Two-thirds of the phospholipase A2 was found in the particulate fractions.     

Chitin biosynthesis in protoplasts and subcellular fractions of Aspergillus fumigatus.

The biosynthesis of chitin has been obtained in broken mycelia and protoplasts of the fungus Aspergillus fumigatus. The specific activity of chitin synthase (EC 2.4.1.16) in a membrane preparation from protoplasts derived from the hyphal tips of A. fumigatus was 26.8-fold greater than that of the chitin synthase in broken mycelia, indicating that the active chitin synthase is located primarily in a membrane-bound site at the hyphal tip. Polyoxin D was a potent competitive inhibitor of the enzyme, having Ki 5.2 +/- 0.8 micron with respect to the natural substrate UDP-N-acetyl-D-glucosamine, which has Km 1.58 mM.     

Distribution and metabolism of glycoproteins and glycosaminoglycans in subcellular fractions of brain.

The distribution, carbohydrate composition, and metabolism of glycoproteins have been studied in mitochondria, microsomes, axons, and whole rat brain, as well as in various synaptosomal subfractions, including the soluble protein, mitochondria, and synaptic membranes. Approximately 90% of the brain glycoproteins occur in the particulate fraction, and they are present in particularly high amounts in synaptic and microsomal membranes, where the concentration of glycoprotein carbohydrate is 2-3% of the lipid-free dry weight. Treatment of purified synaptic membranes with 0.2% Triton X-100 extracted 70% of the glycoprotein carbohydrate but only 35% of the lipid-free protein residue, and the resulting synaptic membrane subfractions differed significantly in carbohydrate composition. The glycoproteins which are not extracted by Triton X-100 also have a more rapid turnover, as indicated by the 80-155% higher specific activity of hexosamine and sialic acid 1 day after labeling with [3H]glucosamine in vivo. The specific activity of sialic acid in the synaptosomal soluble glycoproteins 2 hr after labeling was greater than 100 times that of the synaptosomal particulate fraction, whereas the difference in hexosamine specific activity in these two fractions was only twofold, and by 22 hr there was little or no difference in the specific activities of sialic acid and hexosamine in synaptosomal soluble as compared to membrane glycoproteins. These data indicate that sialic acid may be added locally to synaptosomal soluble glycoproteins before there is significant labeling of nerve ending glycoproteins by axoplasmic transport. Fifty to sixty percent of the hyaluronic acid and heparan sulfate of brain is located in the various membranes comprising the microsomal fraction, whereas half of the chondroitin sulfate is soluble and only one-third is in microsomal membranes. When microsomes are subfractionated on a discontinuous density gradient over half of the hyaluronic acid and chondroitin sulfate are found in membranes with a density less than that of 0.5 M sucrose (representing a six- to sevenfold enrichment over their concentrations in the membranes applied to the gradient), whereas half of the heparan sulfate is present in membranes with a density greater than that of 0.8 M.     

Distribution of enzyme activities in subcellular fractions of bovine retina.

Centrifugation of homogenates of bovine retinas to isopycnic equilibrium in sucrose density gradients yielded three partially overlapping bands of particles which were, in the order of increasing density: (a) photoreceptor cell (rod) outer segments; (b) plasma membranes, lysosomes, and large fragments of endoplasmic reticulum; and (c) mitochondria. The only enzyme activity investigated which had a peak coinciding only with outer segment fractions was guanylate cyclase. Enzyme activities with peaks in both the outer segment and denser fractions included 5'-nucleotidase and cyclic GMP phosphodiesterase. Enzyme activities with peaks only in the denser fractions included sodium and potassium ion-activated ATPase ((Na+ + K+)-ATPase), adenylate cyclase, cyclic AMP phosphodiesterase, beta-glucosidase, beta-galactosidase, and succinate-dependent cytochrome c reductase. These results suggest that some of the activities once thought to be present in rod outer segments are actually present in particles from elsewhere in the retina which contaminate rod outer segment preparations.     

Detection of low molecular mass GTP-binding proteins in chromaffin granules and other subcellular fractions of chromaffin cells

A homogenate of purified chromaffin cells was fractionated, after removal of the nuclear fraction, by sucrose density gradient ultracentrifugation. The presence and subcellular localization of low molecular mass GTP-binding proteins was explored by incubation of blots of proteins from different subcellular fractions with [alpha-32P]GTP in the presence of Mg2+. The fractions enriched in intact chromaffin granule markers, i.e. catecholamines, chromogranin A, chromogranin B and cytochrome b-561 were also enriched in labelled GTP-binding proteins. Two major labelled components of 23 and 29 kDa were rapidly detected by autoradiography. Traces of 26 and 27 kDa components were also present. These components were detectable in both plasma and granule membranes. In addition to these components, the cytosolic fraction contained another GTP-binding protein of about 20 kDa. Binding of [alpha-32P]GTP was specific and dependent on Mg2+. By analogy to the findings reported in non-mammalian systems, the observations described here suggest the involvement of low molecular mass GTP-binding proteins in the chromaffin cell secretory process.     

Amount and time course of ingestion of plant subcellular fractions by two thrips and one reference mite species

Thrips are known as cell-feeding sucking pests, but little information is available on which and how much of each subcellular fraction they ingest. In this study, the ingested amounts of nuclei and plastids in two representative thrips species (the western flower thrips, Frankliniella occidentalis, and the garden thrips, Frankliniella intonsa) along with two reference sucking pests (the two-spotted spider mite, Tetranychus urticae, a cell feeder, and the brown planthopper, Nilaparvata lugens, a sap feeder) were quantified by quantitative PCR using phytoene desaturase and rubisco as respective marker genes following feeding. The ingested amounts of plastids were significantly greater than those of nuclei in the thrips and mite species. In the thrips species, however, the fold differences in ingested amount between the two fractions were substantially lower than their original ratio in intact plant cells, suggesting that thrips ingest nuclei more selectively than plastids. Unlike the thrips species, the ratio between nuclei and plastids increased in T. urticae. In contrast to these cell-feeding insects, no subcellular fraction was detected in N. lugens. These findings suggest that transgenic expression of foreign hairpin RNA in the nucleus would deliver a substantial amount of target molecules to cell-feeding sucking pests, but not sap-feeding pests, when employing ingestion RNA interference-based control strategies.     

Control by TSH of protein turnover in thyroid subcellular fractions.

The action of TSH on protein turnover in various subcellular fractions has been investigated in dog thyroid slices incubated in vitro. The results suggest a general inhibition by TSH of protein catabolism. Using double labeline (3/ and 14C) of the proteins, an increase of the disappearance of some labeled material from the microsomal fraction in the presence of TSH has been observed. The protein nature of this material has been established by testing its susceptibility to hydrolysis by trypsin. The fact that the microsomal pellet had to be treated by triton X 100 before hydrolysis by trypsin could occur, suggests that the material is probably enclosed in, or protected by membrane vesicles. Its high molecular weight and its ability to be immunoprecipitated by an antithyroglobulin serum suggest that the microsomal protein, the disappearance of which is stimulated by TSH, is thyroglobulin or one of its subunits. It is suggested that our results reflect the acceleration by TSH of the vectorial transfer of thyroglobulin through the membranes of the endoplasmic reticulum to the colloid space.