Altered actin and immunoglobulin C mu expression in nitrogen mustard-resistant human Burkitt lymphoma cells.

Raji-HN2 is a B cell lymphoma (Burkitt lymphoma) line that was made resistant to nitrogen mustard. The drug-resistant phenotype was accompanied by changes in gene expression. The expression of four unrelated genes was examined by Northern blot analysis.

Evidence for the participation of topoisomerases I and II in cadmium-induced metallothionein expression in Chinese hamster ovary cells.

CHO-Cdr20 cells are 10-20 times more resistant to killing by cadmium than the parental CHO cells. Resistance has been linked to amplification of the metallothionein genes MT-I and MT-II and their coordinate induction by cadmium and other toxic metals. We studied the roles of the nuclear enzymes topoisomerase I and topoisomerase II in Cd-induced expression of MT-II.

Solubilization of rat liver vasopressin receptors as a complex with a guanine-nucleotide-binding protein and phosphoinositide-specific phospholipase C.

Vasopressin V1 receptors were solubilized from rat liver plasma membranes with the detergent lysophosphatidylcholine. [[3H]Arginine]vasopressin (AVP) binding to the solubilized preparations was specific and saturable, with a dissociation constant of 0.6 nM.

Mammalian phosphoinositide-specific phospholipase C isoenzymes.

Procaryotic and eucaryotic cells have evolved multiple pathways for communication with their external environment. The inositol 1,4,5-trisphosphate/diacylglycerol second messenger system is an example of such a signal transduction pathway which is present in multicellular eucaryotic organisms. Binding of an agonist to a specific cell surface receptor promotes rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate.

Multiple (alpha-NH-ubiquitin)protein endoproteases in cells.

Ubiquitin is encoded as a variable, spacerless repeat of the gene terminating with an additional amino acid or as a gene coding for a single ubiquitin with a carboxyl-terminal extension of 52 to 80 amino acids. We report the identification and partial purification of enzymes that specifically hydrolyze the peptide bond between ubiquitin-ubiquitin conjugate (Ub-Ubase) or ubiquitin fusion proteins (Ub-Xase). The Ub-Ubase was separated from the Ub-Xase by dye-ligand-Sepharose chromatography.

Genetic regulation of beta 2-adrenergic receptors in 3T3-L1 fibroblasts.

The beta 2-adrenergic receptor from mouse 3T3-L1 cells is up-regulated through genetic mechanisms by glucocorticoids and butyrate. To study the genetic regulation of these receptors, we sequenced a 5 kb region of genomic DNA from 3T3-L1 cells, containing the beta-adrenergic receptor gene and approx. 1.

Purification and characterization of predominant G-protein from bovine lung membranes. Biochemical and immunochemical comparison with Gi1 and Go purified from brain.

The predominant guanine nucleotide-binding protein (G-protein) of bovine lung membranes, termed GL, has been purified and compared biochemically, immunochemically and functionally with Gi and Go purified from rabbit brain. The purified GL appeared to have a similar subunit structure to Gi and Go, being composed of alpha, beta and possibly gamma subunits. On Coomassie Blue-stained SDS/polyacrylamide gels and immunoblots, the alpha subunit of GL (GL alpha) displayed an intermediate mobility (40 kDa) between those of Gi and Go (Gi alpha and Go alpha).

Molecular mechanisms of homologous and heterologous desensitization mediated by vasopressin in smooth muscle cells.

Arginine vasopressin (AVP) has been shown previously to enhance phosphatidylinositol (PI) turnover and mobilize calcium in the rat aortic smooth muscle cell-line (A10; ATCC CRL 1476) via the V1 receptor (Aiyar, N., Nambi, P., Stassen, F.

Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli.

Despite the availability of efficient transcription and translation signals, some heterologous gene products are not adequately expressed when introduced into prokaryotes and eukaryotes. An expression system has been established in Escherichia coli to increase the yield of cloned gene products, where the C terminus of ubiquitin was fused to the N terminus of unstable or poorly expressed proteins. Fusion of ubiquitin to yeast metallothionein or to the alpha subunit of the adenylate cyclase-stimulatory GTP-binding protein increased the yield from undetectable to 20% of the total cellular protein.

Induction of c-fos protein by activation of vasopressin receptors in smooth muscle cells.

Stimulation of vasopressin (V1) receptors of rat aortic smooth muscle cells (A-10, ATCC CRL 1476) results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) with the mobilization of intracellular calcium. When A-10 cells are exposed to arginine vasopressin (AVP), there is an increase in the level of c-fos oncoprotein. The extent of induction of c-fos oncoprotein depends on both the time of exposure of the cells to AVP, reaching a maximum at 60 min after which there is a slow decline, and the concentration of AVP used, with an approximate EC50 of 1 nM which corresponds well with the Kd of vasopressin binding to these receptors.

Gene synthesis, expression, and processing of human ubiquitin carboxyl extension proteins.

In order to study 1) the mechanisms responsible for generating free ubiquitin monomer and 2) the function of ubiquitin carboxyl extension proteins in eukaryotes, we have developed a system for expression of human ubiquitin carboxyl extension proteins in prokaryotic and eukaryotic hosts. When expressed in Saccharomyces cerevisiae, the intact ubiquitin carboxyl extension proteins were rapidly processed to free ubiquitin monomer and extension protein. Furthermore, expression in this host conferred a slow growth phenotype mediated by the extension protein.

The signal transduction system of the leukotriene D4 receptor.

During the past several years, substantial progress in understanding the receptors and signal transduction processes for peptidyl leukotrienes has been reported. Receptors have been identified and characterized, the major steps in the signal transduction pathway have been described, and the genetic and epigenetic regulatory processes have been characterized. Very recent studies have defined the mechanisms by which LTE4 acts as a partial agonist at the LTD4 receptor.

Solubilization of a guanine nucleotide-sensitive form of vasopressin V2 receptors from porcine kidney.

Vasopressin (V2) receptors were solubilized from porcine kidney membranes with the detergent egg lysolecithin. Binding of [3H]vasopressin to the solubilized fraction was rapid, specific, and saturable. The agonist dissociation constants observed in membranes and solubilized fractions were 1.

Ubiquitin function studied by disulfide engineering.

Disulfide engineering was used to probe the role of conformational mobility in ubiquitin-mediated proteolysis. Six genes that encode cysteine-containing mutants of ubiquitin were constructed, expressed in Escherichia coli and the proteins purified. Single cysteine-containing mutants and a 4/14 disulfide were active in degradation of a substrate protein in vitro, while the 4/66 disulfide, which cross-links the NH2- and COOH-terminal strands of the protein, was only 20-30% active.

Homologous and heterologous desensitization mediated by vasopressin in smooth muscle cells.

Prolonged exposure of A-10 cells to Arginine Vasopressin (AVP) resulted in the following responses: (a) loss of vasopressin receptors from the cell surface (30-40%), (b) increased basal levels of inositol and inositol monophosphate, (c) decreased inositol di- and trisphosphate production and decreased intracellular calcium release in response to a second challenge with AVP, (d) attenuation of AVP-mediated inhibition of isoproterenol-stimulated cAMP and ANF-stimulated cGMP accumulation and (e) attenuation of thrombin and ATP-mediated increase in inositol di- and trisphosphate accumulation and intracellular calcium release. All the above responses depended on the time of exposure of the cells to AVP with the responses being attenuated as early as 5-10 min of exposure to AVP. The desensitization also depended on the concentration of AVP used with 50% of maximal desensitization for each response being observed at 5 nM of AVP.

Immunochemical comparison of pertussis toxin substrates in brain and peripheral tissues.

The tissue distribution of pertussis toxin-sensitive GTP-binding proteins was examined using specific antibodies raised against the purified alpha-subunit of G0 from bovine brain or against synthetic peptides predicted from cDNAs for distinct Gi subtypes. GTP-binding proteins were partially purified from membrane fractions prepared from rabbit tissues including brain, heart, liver, lung, erythrocytes and neutrophils. Brain contained both G0 and Gi1.

Phorbol 12-myristate 13-acetate inhibition of leukotriene D4-induced signal transduction was rapidly reversed by staurosporine.

Activation of leukotriene D4 receptors results in phospholipase C-mediated breakdown of phosphatidylinositol and increases in intracellular Ca2+ in U-937 cells. Treatment (10 min) with phorbol 12-myristate 13-acetate blocked leukotriene D4-induced phosphatidylinositol metabolism and Ca2+ mobilization (IC50 = 0.2 nM).

Ubiquitin-metallothionein fusion protein expression in yeast. A genetic approach for analysis of ubiquitin functions.

We have established a Saccharomyces cerevisiae genetic system that expresses the fusion protein ubiquitin-metallothionein. We have evaluated the effects of amino-terminal ubiquitination of metallothionein on the stability and function of metallothionein. The fusion protein of wild type ubiquitin and metallothionein was rapidly processed in vivo to release free ubiquitin and metallothionein.

Leukotriene D4 receptor-mediated phosphoinositol hydrolysis and calcium mobilization in rat basophilic leukemic cells.

A basophilic leukemic cell line from rat (RBL-1) was used to characterize leukotriene D4 (LTD4) receptor-mediated biochemical and pharmacological effects. [3H]LTD4 binding to the plasma membrane enriched preparation was stereo-selective, specific and saturable. Sodium ions and guanine nucleotides specifically regulated [3H]LTD4 binding to the membrane receptors.

A murine model to evaluate the ability of in vitro clonogenic assays to predict the response to tumors in vivo.

The use of the human tumor cloning assay as a predictor of clinical response of human tumors to drugs is predicated on the hypothesis that the in vivo response of a tumor to a drug can be correlated with the in vitro response of cells derived from the tumor. To test this hypothesis, we utilized a murine tumor model in which the in vivo and in vitro responses of a tumor can be accurately and reproducibly compared. Drug activity was assessed in P388 leukemia with the standard in vivo antitumor assay (i.

Subcellular localization of leukotriene D4 receptors in sheep tracheal smooth muscle.

The distribution of [3H]leukotriene D4 [( 3H]LTD4) receptors in subcellular membrane fractions obtained from sheep tracheal smooth muscle was studied. Using differential centrifugation and discontinuous sucrose density gradient centrifugation, the subcellular membranes were separated into six fractions. The [3H]LTD4 receptor distribution profile in these fractions correlated with markers for the plasma membrane (5'-nucleotidase and alkaline phosphodiesterase) and did not correlate with markers for the mitochondria (cytochrome c oxidase and succinate-dependent cytochrome c reductase).

Leukotriene D4-induced homologous desensitization in basal and differentiated U-937 cells: characterization with the partial agonist leukotriene E4 and assessment of receptor reserve.

U-937 cells express receptors for leukotriene D4, and LTD4 stimulates receptor-mediated Ca++ mobilization with an EC50 of 5 nM. In these cells, LTE4 produces Ca++ mobilization with an EC50 of 500 nM and a maximum response that is 15 to 20% of the LTD4 response. LTE4 was shown to be a partial agonist, with a Kp of 400 nM and a linear relationship between effect and receptor occupancy, and was used as a tool to study LTD4-induced desensitization.