Hormone-regulated expression of cellular rasH oncogene in mammary carcinomas in rats

Differential gene expression has been observed in hormone-dependent rat mammary carcinomas during their growth and regression. A 22K MW protein, a prominent in vitro translation product of the growing as compared to the regressing tumor, was identified as the c-rasH-21,000-dalton transforming protein (p21) using a monoclonal antibody that reacts specifically with Harvey-related p21 species. The amount of p21-translated protein sharply decreased in the translation products of the regressing tumors within 6 hours post ovariectomy or dibutyryl cyclic AMP treatment. The results show that an enhanced expression of the c-rasH oncogene is associated with hormone-dependent growth of mammary carcinomas in vivo and that suppression of this oncogene precedes the tumor regression induced by either hormone withdrawal or dibutyryl cyclic AMP treatment.     

Inhibition of parathormone-stimulated bone resorption by type I interferon

The effect of Type I interferon on bone resorption was studied by measuring its effect on parathormone-stimulated calcium release from neonatal murine calvaria in vitro. A pure human recombinant leukocyte interferon hybrid of the A and D subtypes was used, which has high antiviral activity on mouse cells. Calcium release was inhibited in a dose dependent fashion with 50% inhibition at about 10(-10) M or 600 U/ml, and the inhibition was reversible. The presence of interferon was required before or during the activation phase of the resorptive response, when the formation of osteoclasts from precursor cells would occur. When added to actively resorbing bone it had no effect. The data suggest that Type I interferon can inhibit the parathormone-regulated development of active osteoclasts, possibly by inhibiting osteoclast precursor differentiation.     

Preparation of isolated liver endothelial cells and Kupffer cells in high yield by means of an enterotoxin

A new method for preparing non-parenchymal rat liver cells (NPC) is described. The liver cell suspension, prepared by perfusing the liver with collagenase, was treated with enterotoxin from Clostridium perfringens for 15 min. The enterotoxin made the parenchymal cells leaky, and these cells could be separated from the NPC by centrifugation in a solution containing Nycodenz (20%, w/v). During the centrifugation, the NPC floated, while the parenchymal cells sedimented. The yield of NPC per liver (200 g rat) was about 250 X 10(6) cells. The NPC were further separated into endothelial cells, Kupffer cells and stellate cells by centrifugal elutriation. This method was particularly useful for preparing endothelial cells in high yield (100 X 10(6) cells per liver). Intravenously injected formaldehyde-treated albumin was selectively taken up by the endothelial cells. Isolated endothelial cells in suspension as well as in surface culture maintained their ability to endocytose this ligand.     

Monovalent carboxylic ionophores inhibit transport of carbamoyl-phosphate synthetase I into mitochondria in Reuber hepatoma H-35 cells and cause accumulation of enzyme precursor

Transport of the precursor for carbamoyl-phosphate synthetase I into mitochondria in Reuber hepatoma H-35 cells was inhibited by adding monensin or nigericin to the culture medium at a concentration of 0.5 microM, and the enzyme precursor accumulated, mainly in the cytosolic fraction. Accumulated precursor was degraded slowly with a half-life of more than 16 min. Valinomycin, nonactin, A23187, X-537A (lasalocid), bromo-lasalocid, and carbonyl cyanide m-chlorophenylhydrazone did not exhibit these effects at concentrations at which they did not inhibit protein synthesis of the cells.     

Assessment of a model for intron RNA secondary structure relevant to RNA self-splicing--a review

A widespread class of introns is characterized by a particular RNA secondary structure, based upon four conserved nucleotide sequences. Among such "class I" introns are found the majority of introns in fungal mitochondrial genes and the self-splicing intron of the large ribosomal RNA of several species of Tetrahymena. A model of the RNA secondary structure, which must underlie the self-splicing activity, is here evaluated in the light of data on 16 further introns. The main body or "core structure" of the intron always consists of the base-paired regions P3 to P9 with the associated single-stranded loops, with P2 present also in most cases. Two minority sub-classes of core structure occur, one of which is typical of introns in fungal ribosomal RNA. Introns in which the core structure is close to the 5' splice site all have an internal guide sequence (IGS) which can pair with exon sequences adjacent to the 5' and 3' splice sites to align them precisely, as proposed by Davies et al. [Nature 300 (1982) 719-724]. In these cases, the internal guide model allows us to predict correctly the exact location of splice sites. All other introns probably use other mechanisms of alignment. This analysis provides strong support for the RNA splicing model which we have developed.     

Inhibition of aggregation of human platelets by 8,15-dihydroperoxides of 5,9,11,13-eicosatetraenoic and 9,11,13-eicosatrienoic acids

The dihydroperoxy derivative of arachidonic acid, 8,15-dihydroperoxy-5,9,11,13-eicosatetraenoic acid, at , is an effective inhibitor of the aggregation of human blood platelets, induced by ADP, epinephrine and collagen. The nature of the effect is time dependent. If ADP is added 60 sec after the inhibitor, the first phase of aggregation occurs but the second phase is suppressed. When the time is prolonged to 240 sec, the first phase of aggregation is almost completely eliminated. The corresponding dihydroxy derivative is without effect on aggregation. The corresponding dihydroperoxy derivative obtained from 8,11,14-eicosatrienoic acid is also an effective inhibitor.     

Mitochondrial development during myogenesis.

The enzymatic and ultrastructural pattern of mitochondrial differentiation was investigated during myogenesis. Succinate cytochrome C reductase (SCR), a mitochondrial enzyme complex, increased in activity in developing chick thigh muscle in vivo and in vitro. SCR increase in vitro occurred subsequent to myoblast fusion and correlated with the period of increasing creatine phosphokinase (CPK) activity. Fusion-arrest and Ca²⁺-reversal experiments indicated an apparent coordination between CPK and SCR enzymatic increases and fusion. Analysis of SCR activity in fibroblast cultures suggested that the enzymatic increases observed in differentiating muscle cultures reflected myocyte differentiation, rather than fibroblast contamination or a unique property of the tissue culture environment. Morphological transitions in the myogenic mitochondria were temporally correlated with increased SCR activity. During myogenesis, the mitochondria enlarged in length and volume, exhibited an increase in matrix density, oriented in parallel to the long axis of the myofibrils, and contained increased numbers of parallel cristae. Many mitochondria in fusion-inhibited muscle cultures resembled those found in prefusion myoblasts, although mature mitochondria were observed in some fusion-blocked cells. Quantitative stereological analyses of these mitochondrial changes parallel the biochemical data and suggest that ultrastructural and enzymatic changes in the mitochondria are an integral part of myodifferentiation.     

Interaction of peanut agglutinin,a lectin specific for nonreducing terminal d-galactosyl residues,with embryonal carcinoma cells

Peanut agglutinin (PNA), a lectin specific for terminal d-galactosyl residues, was found to react with embryonal carcinoma cells, but not with their differentiated derivatives. Receptors for PNA were detectable at the surface of all cells of the quasinullipotent F9 line and on only 50% of the multipotent PCC3/A/1 line. The fraction of the PCC3/A/1 population which expresses the F9 antigen was found to be included in the subpopulation carrying the PNA receptors. PNA⁺ and PNA^? subpopulations of PCC3/A/1 were separated by a PNA-mediated reversible agglutination of PNA⁺ cells with rabbit erythrocytes. These subpopulations were essentially F9⁺ and F9^?, respectively.     

The polyadenylated RNA of zoospores and growth phase cells of the aquatic fungus,Blastocladiella

The stored poly(A) + RNA from zoospores of the aquatic fungus Blastocladiella emersonii represents 2.5% of the total RNA and has a model MW of 425,000 daltons and an average poly(A) isostich of 32 bases. The poly(A) + RNA also represents 2.5% of the total RNA from early growth phase cells and has a modal MW of 360,000 daltons and an average poly(A) isostich of 38 bases. The poly(A) + RNA from spores and 2-hr plants contains a structure resistant to RNases T₁, T₂, and A, which can be labeled with ³²PO₄ and which will bind to DBAE-cellulose. These characteristics strongly suggest that both the zoospore poly(A) + RNA and the 2-hr cell poly(A) + RNA are capped at the 5′ end; and, hence, it is unlikely that capping is involved in the control of protein synthesis during germination.Approximately 80% of the poly(A) + RNA of the spore is located in the membrane-enclosed ribosomal nuclear cap, and more than 90% of the poly(A) + RNA within the cap is found in the 80S monoribosome and heavier fractions.Synthesis of new poly(A) + RNA occurs very early during zoospore germination, and the labeled poly(A) + RNA rapidly enters the newly organized polysomes. The labeling data for early germination also suggest that cytoplasmic polyadenylation occurs.