Showing papers by "National Jewish Health published in 1985"

The small-subunit ribosomal RNA gene sequences from the hypotrichous ciliates Oxytricha nova and Stylonychia pustulata.

Abstract: We have determined the complete nucleotide sequence of the small-subunit ribosomal RNA genes for the ciliate protozoans Stylonychia pustulata and Oxytricha nova. The sequences are homologous and sufficiently similar that these organisms must be closely related. In a phylogeny inferred from comparisons of several eukaryotic small-subunit ribosomal RNAs, the divergence of the ciliates from the eukaryotic line of descent is seen to coincide with the radiation of the plants, the animals, and the fungi. This radiation is preceded by the divergence of the slime mold, Dictyostelium discoideum.

The role of glutathione in lymphocyte activation. I. Comparison of inhibitory effects of buthionine sulfoximine and 2-cyclohexene-1-one by nuclear size transformation.

Abstract: The role of glutathione (GSH) in lectin-induced lymphocyte activation can be studied by quantitating lectin-induced nuclear size transformation in the presence of variable degrees of GSH depletion. Buthionine sulfoximine (BSO) inhibits intracellular GSH synthesis by inhibition of the enzyme gamma-glutamyl-cysteine synthetase. By combining endogenous GSH depletion in cell cultures with BSO-induced inhibition of GSH synthesis, lectin-induced lymphocyte activation can be studied at various concentrations of soluble intracellular GSH. With this approach, the percentage of lymphocytes undergoing a nuclear size transformation is minimally affected despite depletion of soluble intracellular GSH to 0.27 nmol/10(7) cells (PBL), which represents approximately 95% depletion of intracellular GSH. When soluble intracellular GSH is depleted to undetectable levels (less than 0.10 nmol/10(7) cells) there is a 10 to 12% reduction in the number of cell nuclei transformed. However, in all BSO-pretreated cultures the lectin-induced nuclear size transformation is intermediate between resting and blast-transformed lymphocytes, suggesting only partial (or aborted) activation. The partial activation response observed in BSO-pretreated cultures may be due to mobilization of the protein-bound pool of GSH, which is relatively resistant to depletion by BSO. That the inhibition of full blast transformation is truly due to GSH depletion was proven by experiments in which GSH was repleted exogenously and a full blast transformation was restored. The results of previous work in our laboratory had shown that the sulfhydryl-reactive agent 2-cyclohexene-1-one (2-CHX) was a potent inhibitor of activation at soluble intracellular GSH concentrations well above 0.27 nmol/10(7) PBL. In the present study, the dose-dependent inhibition of activation by 2-CHX was confirmed, but it was shown that the degree of inhibition caused by 2-CHX could be at least partially dissociated from the level of intracellular GSH present at the time of lectin addition and that the inhibitory potential of 2-CHX exceeded that of BSO at comparable levels of soluble intracellular GSH. Thus, the inhibitory properties of 2-CHX cannot be accounted for solely on the basis of GSH depletion.

Changes in cyclic adenosine 3':5'-monophosphate-dependent protein kinases during the progression of urethan-induced mouse lung tumors.

Abstract: The cyclic adenosine 3′:5′-monophosphate (cAMP)-dependent protein kinases in lung adenomas are functionally different from those of normal lung. The relevance of this change to neoplastic conversion was examined by comparing tumor kinases with those obtained from the normal cell of origin and by studying the kinases at different stages of tumor growth. Lung tumors were collected from A strain mice at different times after a single injection of urethan. These tumors are predominantly of alveolar type two cell origin, and cAMP-binding proteins in extracts from isolated type two cells and from lung adenomas at various stages of tumor progression were compared. Both the incorporation of the cAMP photoaffinity analogue, cyclic 8-azidoadenosine 3′:5′-[ 32 P]monophosphate (8-N 3 -[ 32 P]cAMP), into the regulatory subunits of the type I (R I ) and type II (R II ) cAMP-dependent protein kinases and the autophosphorylation of R II were similar in extracts from whole normal lung and from type two cells. Altered protein kinases are thus not characteristic of normal type two cells. Lung tumors showed a decrease in photodetectable R II which correlated in degree with tumor size and extent of anaplasticity. This decreased R II photolabeling during tumor growth was associated with increased R II autophosphorylation. In contrast, decreased R II photolabeling in extracts from neonatal lung is accompanied by a substantial decrease in R II autophosphorylation. The characteristics of R II during normal development thus clearly differ from those during neoplastic development. An increase in the amount of an M r 37,000 proteolytic fragment derived from R-subunits was also noted as a function of tumor progression. DEAE-cellulose chromatography of tumor cytosol showed that the increase in the amount of M r 37,000 protein was accompanied by increased subunit dissociation of the type I isozyme. The dissociated R I subunit has been shown to be more sensitive to cleavage by a Ca 2+ -dependent neutral protease than when R I was in the holoenzyme form. This protease is present in both normal lung and lung adenomas, and its activity increases during the later stages of tumor progression. A comparison of cAMP binding and the light-induced covalent incorporation of 8-N 3 -[ 32 P]cAMP showed that, for both R I and R II , photoincorporation was about 75% as efficient as noncovalent binding. In contrast, although the M r 37,000 fragment can be photolabeled with low concentrations of 8-N 3 -[ 32 P]cAMP, noncovalent cAMP binding to the endogenous M r 37,000 fragment could not be demonstrated with a standard filtration assay. Such altered cAMP binding characteristics following Ca 2+ -dependent proteolysis of R-subunits would allow significant synergism of cAMP and Ca 2+ on protein kinase activation.