Effect of thyroid hormone on the abundance of Na,K-adenosine triphosphatase alpha-subunit messenger ribonucleic acid

The effects of thyroid hormone on Na,K-ATPase alpha-subunit mRNA (mRNA alpha) content and Na,K-ATPase activity were measured in renal cortex, heart, and cerebrum of hypothyroid rats 24 and 72 h after injection of diluent or T3. Use of a cDNA probe complementary to rat brain mRNA alpha in Northern blot analysis revealed a single 26-27 S band in RNA isolated from these three tissues regardless of thyroid status. Tissue mRNA alpha content was estimated by dot blot analysis of whole cell extracts and isolated total RNA. Injection of T3 augmented mRNA alpha content by 2.1- to 2.5-fold in kidney cortex and myocardium at 24 h. After three daily injections of T3, the increases in mRNA alpha were evident despite a global increase in RNA content associated with hypertrophy of these target tissues. Furthermore, the increases in abundance of mRNA alpha after 72 h of T3 treatment correlated with enhancement of Na,K-ATPase activity. In contrast, both mRNA alpha and enzyme activity were invariant in the cerebrum. These data suggest that T3-induced augmentation of Na,K-ATPase activity is mediated, at least in part, by increased mRNA alpha content in target tissues.     

Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit

We have used a gene transfer system to investigate the relationship between expression of the rat Na,K-ATPase alpha 1 subunit gene and ouabain-resistant Na,K-ATPase activity. A cDNA clone encoding the entire rat Na,K-ATPase alpha 1 subunit was inserted into the expression vector pSV2neo. This construct (pSV2 alpha 1) conferred resistance to 100 microM ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of transfected clones revealed the presence of both rat-specific and endogenous Na,K-ATPase alpha 1 subunit DNA and mRNA sequences. A single form of highly ouabain-sensitive 86Rb+ uptake was detected in CV-1 cells, whereas two distinct classes of ouabain-inhibitable uptake were observed in transfectants. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K-ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Examination of the ouabain-sensitive, sodium-dependent ATPase activity of the transfectants also revealed a low affinity component of Na,K-ATPase activity characteristic of the rodent kidney enzyme. These results suggest that expression of the rat alpha 1 subunit gene is directly responsible for ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells.     

The determination in saliva of IgA antibodies to Shigella ribosomes for the diagnosis of dysentery

The levels of antiribosomal antibodies to Shigella ribosomes in serum and saliva samples from 38 dysentery patients (15 S. sonnei cases and 23 S. flexneri cases), 14 patients with salmonellosis and 136 healthy adults were determined in ELISA with ribosomes from S. sonnei R-mutant used as solid-phase antigen. High levels of "normal" antiribosomal IgA, IgG and IgM antibodies were revealed in the sera of healthy persons while the level of salivary IgA antibodies was very low. In dysentery infection no increase in the levels of serum IgG and IgM antibodies and only a slight increase in the level of IgA antibodies were revealed. Local immune response was manifested by the early (on days 2-4 from the onset of infection) and significant augmentation (12- to 16-fold) of salivary antiribosomal IgA antibodies. An increase in the level of these antibodies was registered in 95-100% of dysentery patients but not in patients with salmonellosis, which made it possible to recommend the method for diagnosing shigellosis. Immune response to Shigella ribosomal antigens, in contrast to the response induced by Shigella O-antigen, is almost exclusively local.     

Growth kinetics as a function of ploidy in diploid, tetraploid, and octaploid smooth muscle cells derived from the normal rat aorta

The smooth muscle cell population in major arteries of humans and experimental animals is heterogeneous with regard to cellular DNA content. A proportion of cells has polyploid DNA content and this proportion increases with normal aging and with hypertension. We have isolated pure populations of rat aortic smooth muscle cells containing 2C, 4C, and 8C DNA content by cloning of cultures of cells previously subjected to flow cytometric cell sorting. Karyologic analysis of these clonal populations revealed them to be pure diploid, tetraploid, and octaploid populations, respectively, containing 2N (= 42), 4N, and 8N chromosomes. Cell attachment area and nuclear size appeared to increase with the level of ploidy. Studies of the proliferative characteristics of the cells revealed that the growth rate and ultimate cell densities achieved decreased as the ploidy level increased. The intrinsic cellular radiosensitivity of these clones did not vary with ploidy. Increased smooth muscle cell ploidy is, therefore, associated with a decreased rate of proliferation. The emergence of smooth muscle cells with polyploid DNA content under normal and pathologic conditions is probably due to mitotic polyploidization without net cell proliferation and may be related to the need for expression of differentiated functions.     

Insulin-stimulated protein tyrosine phosphorylation in intact cells evaluated by giant two-dimensional gel electrophoresis

We have studied the insulin-stimulated phosphorylation of proteins in NIH 3T3 cells expressing high numbers of human insulin receptors (HIR 3.5 cells) using the technique of giant two-dimensional gel electrophoresis. In serum-deprived cells, insulin stimulated the phosphorylation of more than 25 proteins; all but two of these were also phosphorylated in response to 15% (v/v) fetal bovine serum, which also stimulated the phosphorylation of additional proteins thought to be direct substrates for protein kinase C. In cells pretreated insulin specifically stimulated the phosphorylation insulin specifically stimulated the phosphorylation of at least 26 predominantly cytosolic proteins, only one of which was observed in insulin-treated cells not exposed to phenylarsine oxide. Serum was without effect in cells pretreated with phenylarsine oxide. In phenylarsine oxide-pretreated cells, phosphoamino acid analysis of 10 of the most highly labeled insulin-stimulated phosphoproteins showed that all 10 were labeled predominantly or exclusively on tyrosine residues. The phosphorylation of several of these could be stimulated in vitro by the addition of insulin to a detergent extract of cells in the presence of Mn2+ and ATP. In general, the insulin-stimulated phosphorylations observed in the presence of phenylarsine oxide were more rapid than those observed in its absence. Finally, a variety of other growth factors and mitogens did not stimulate any of the insulin-stimulated phosphorylations in the presence of phenylarsine oxide. Thus, the use of this inhibitor apparently unmasked a number of novel insulin-specific protein phosphorylations that were ordinarily undetectable. We suggest that at least some of these proteins may be direct substrates for the insulin receptor protein tyrosine kinase and may play significant roles in insulin action.     

Developmental program of murine erythroleukemia cells. Effect of the inhibition of protein synthesis

The relationship between protein synthesis and commitment to terminal erythroid differentiation by dimethylsulfoxide-treated murine erythroleukemia (MEL) cells has been studied. Treatment with cycloheximide blocks the commitment of MEL cells. The effects of cycloheximide are completely reversible, however. Treatment of MEL cells before commitment delays commitment for a period of time equal to the length of inhibitor treatment. Puromycin exerts a similar effect on the commitment of MEL cells. These results indicate that there is a continuous requirement for protein synthesis before the commitment event.     

Significance of the cell cycle in commitment of murine erythroleukemia cells to erythroid differentiation.

The relationship between differentiation of murine erythroleukemia cells (MEL) induced by DMSO and the cell division cycle has been analyzed. We demonstrate that incubation in the presence of DMSO increases the length of the G1 phase of the cell cycle. A method of synchronization of MEL cells by unit gravity sedimentation has been developed and characterized. Using this method, a series of synchronized cell populations covering the entire cell division cycle can be generated simultaneously. Cells synchronized by this technique were challenged with DMSO and analyzed for kinetics of commitment to the differentiation program. Our results indicate that populations of cells in G1 or G2 at the time of addition of inducer give rise to a greater proportion of committed cells than an unfractionated population, while cells in S phase result in a lower percentage of committed cells than the unfractionated population when cultured in DMSO.     

NMR investigation of the interaction of mithramycin A with d(ACCCGGGT)2.

The binding of mithramycin A to d(ACCCGGGT)2 has been investigated by one- and two-dimensional 1H NMR spectroscopy. Titration of the drug into the octamer solution results in loss of the oligonucleotide C2 symmetry at stoichiometric ratios less than 4 drug molecules per duplex. However, at a ratio of 4:1 (drug/duplex), the C2 symmetry of the oligonucleotide is restored. From these data it is evident that more than one complex forms at ratios less than 4:1 while only one complex predominates at the ratio 4:1. This is the first report of a DNA octamer which binds 4 large drug molecules. These results are compared to those we have recently reported for mithramycin binding to d(ATGCAT)2, where only a single, bound complex is observed, with a stoichiometry of 2:1.     

Erasure of the memory response in MEL cells

When MEL cells are reexposed to DMSO after an interruption in inducer treatment, they can initiate commitment to differentiation without the lag period observed after the primary exposure to inducer. This property is known as memory. Here we have employed metabolic inhibitors to analyze the basis of the memory response. Treatment of cells with cycloheximide or cordycepin during the inducer withdrawal period causes memory erasure. Cells must recapitulate an entire lag period upon reexposure to DMSO. The memory response is maintained, however, if cells are treated with metabolic inhibitors in the presence of DMSO. Our results suggest that the capacity of MEL cells for memory requires the synthesis of cell components which are normally stable in the absence of DMSO. Experiments involving reciprocal shifts between two different inhibitors have been performed. Evidence is presented that the process leading to the initiation of commitment is composed of at least three components acting in sequence.     

Memory of MEL cells to a previous exposure to inducer.

The mechanism of commitment of murine erythroleukemia (MEL) cells to terminal differentiation has been examined. Before a significant proportion of cells becomes committed, a lag period of at least 9 hr of exposure to inducer is observed. Cells withdrawn from inducer can reinitiate commitment without a lag when reexposed. The proportion of committed cells in a culture discontinuously exposed to inducer is identical to that in a continuously exposed culture even if withdrawal from inducer lasts for 18 hr. The ability to tolerate an interruption in the exposure has been termed "memory." The memory of a previous exposure to inducer is complete up to 18 hr. It is partially erased after 36 hr and completely erased after 72 hr. The length of time the memory persists is not affected by the length of the initial exposure to inducer. These results suggest that a cellular component necessary for the commitment event accumulates in response to inducer and that this component has a decay time on the order of 10 hr.