Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Toxin B from Clostridium difficile induces typical morphological changes of cultured cells consisting of rounding up and arborization, which are associated with a dramatic disruption of microfilaments. In this study, we show that toxin L, a cytotoxin produced by bacterial strain Clostridium sordellii, has similar effects on cultured cells including the redistribution of F-actin and of the adhesion plaque protein vinculin. It has been assumed that the mechanisms involved in cytopathic effects of toxin B are related to the function of an unidentified component that regulates the organization of the actin cytoskeleton. We demonstrate that the treatment of cultured astrocytes with toxin B or toxin L alters the incorporation of inorganic phosphate into several proteins. Immunoblot analysis revealed that one of these proteins is tropomyosin. Since tropomyosin stabilizes microfilaments and inhibits the severing activity of gelsolin, the toxin-induced phosphorylation may counteract this inhibition resulting in severing of microfilaments and capping of short filaments. A decrease in the radioactivity associated with intermediate filament protein vimentin was also detected using a monoclonal antibody which specifically recognizes a phosphorylated epitope of vimentin. Since vimentin is an in vivo substrate for various protein kinases, these data are in favor of broad effects of these toxins. Direct measurement of protein kinase C in cells exposed to toxin B or to toxin L did not reveal a significant change in protein kinase C activity. Furthermore, treatments with toxins do not increase cAMP levels, suggesting that toxins do not activate protein kinase A. Although further studies are required to determine the primary target site for the clostridial cytotoxin B and L, our results show that they provoke the alteration in the phosphorylation of cellular proteins.
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PMID:Phosphorylation of cellular proteins in response to treatment with Clostridium difficile toxin B and Clostridium sordellii toxin L. 172 54

Both the secretory and cytotoxic activity of natural killer (NK) cells are known to be regulated by such cytokines as interleukin-2 (IL-2) and interferon-gamma (IFN-gamma). In the present study we have used the reverse hemolytic plaque assay to investigate either the direct effects of the protein kinase activator, phorbol myristate acetate (PMA), or exposure to recombinant human interleukins 2, 4, and 6 (IL-2, IL-4, and IL-6) tumour necrosis factor alpha (TNF-alpha) and basic fibroblast growth factor (bFGF) on the release of IFN-gamma by individual, immunoidentified NK cells isolated from peripheral blood. This sensitive immunoassay was adapted and coupled with immunocytochemistry not only to immunophenotype and enumerate cells secreting IFN-gamma in a given cell population, but also to quantify the amount of this cytokine released per individual cell. These studies have confirmed mononuclear cells with the morphology of large granular lymphocytes and the immunophenotype of CD3-/CD16+ NK cells to be the predominant source of spontaneously released IFN-gamma in vitro. In contrast to this, fewer than 2% of the CD3+ T cells secreted detectable levels of this cytokine during the assay, irrespective of the stimulus applied. Whilst TNF-alpha had no significant effect on IFN-gamma release by NK cells, a 6-hr exposure to IL-2 or PMA stimulated an increase in the amount secreted per single cell. Furthermore, bFGF and interleukins 4 and 6 elicited a marked, dose-dependent stimulation of IFN-gamma secretion by this cell type. However, exposure to these cytokines did not alter the number of cells capable of releasing detectable levels of IFN-gamma during the assay. These studies demonstrate that (i) both the spontaneous and stimulated release of IFN-gamma by NK cells can be visualized and quantified at the single-cell level using this sensitive immunoassay, and (ii) bFGF and interleukins 2, 4, and 6, but not TNF-alpha, are potent stimulants of IFN-gamma secretion by CD3-/CD16+ NK cells.
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PMID:Basic fibroblast growth factor and interleukins 4 and 6 stimulate the release of IFN-gamma by individual NK cells. 182 52

Isolation of cDNA clones from lambda gt11 phage libraries by functional screening is limited by the low amount of lacZ-cDNA-encoded fusion protein synthesized in an isolated phage plaque. The amount of specific cDNA-encoded protein can be significantly enhanced by expression in bacterial colonies rather than phage plaques. Escherichia coli was lysogenized with a lambda gt11 cDNA expression library from Dictyostelium discoideum. Bacteria were selected for the presence of the lambda gt11 prophage by elimination of nonlysogenic parental cells with a lambda cI phage. The usefulness of the lysogen library was demonstrated by immuno-screening and functional screening with two different radiolabeled ligands. cDNA clones encoding a well-characterized D. discoideum protein, the regulatory subunit of the cAMP-dependent protein kinase, were isolated by screening the lysogen library with antibodies. Clones encoding this protein could also be identified by functional screening with [3H]cAMP, demonstrating that the limit of detection of positive clones by ligand screening is at least an order of magnitude lower for the lysogen library than for the corresponding phage library. We have subsequently used the lysogen library to isolate cDNA clones encoding calmodulin-binding protein(s) from D. discoideum by functional screening with [125I]calmodulin. For these clones, screening of the corresponding phage library had previously been found unsuccessful.
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PMID:Prophage lambda libraries for isolating cDNA clones by functional screening. 214 39

We previously demonstrated that dexamethasone treatment of L929 cells inhibited plaque formation by vesicular stomatitis virus (VSV), encephalomyocarditis virus, or vaccinia virus. We now have characterized the antiviral effects of glucocorticoids in L929 cells. Dexamethasone did not directly inactivate VSV nor did steroid treatment of L929 cells affect virion adsorption or penetration. The VSV yield in L929 cells treated with dexamethasone for a period of only 4 or 8 hr was decreased by 50% when cells were infected the day following steroid treatment. Treating L929 cells with dexamethasone for a longer period resulted in greater inhibitions of virus synthesis. Interferon activity (less than 5 units/ml) was not detected in L929 cell culture fluids and cell sonicates from steroid-treated cells and the addition of antiserum to murine alpha/beta-interferon had no effect on the ability of dexamethasone to inhibit VSV replication. Dexamethasone treatment of L929 cells did not induce the production of double-stranded RNA-dependent protein kinase but did result in a slight elevation of 2-5A oligoadenylate synthetase activity, two enzymatic activities associated with the antiviral state induced by interferon. However, the elevated 2-5A synthetase activity was not associated with an inhibition of VSV RNA accumulation in dexamethasone-treated L929 cells. By contrast, the synthesis of all five VSV proteins was reduced by 50-75% in dexamethasone-treated L929 cells as early as 4 hr after infection. Thus, the dexamethasone-mediated inhibition of VSV replication in L929 cells is associated with decreased production of VSV structural proteins.
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PMID:Inhibition of vesicular stomatitis virus replication in dexamethasone-treated L929 cells. 215 55

One isoform of the regulatory subunit of type II cAMP-dependent protein kinase (R-II51; Mr = 51,000) and its electrophoretic variants (R-II51.5 and R-II52; Mr = 51,500 and 52,000, respectively) are selectively induced by estradiol and follicle-stimulating hormone (cAMP) in rat ovarian granulosa cells. To ascertain the amino acid sequence of R-II51 and to gain insight into the molecular events regulating the intracellular content of ovarian follicular R-II51, we constructed a lambda gt11 cDNA expression library from poly(A)+ RNA of hormone-primed rat granulosa cells. A 1.5-kilobase (kb) cDNA insert, isolated from a plaque-purified R-II antibody positive bacteriophage clone, selectively bound R-II51 mRNA as demonstrated by analysis of the hybrid-selected translation product. Restriction maps and sequence analyses of the 1.5-kb cDNA insert and of the 1.8- and 2.2-kb cDNA inserts from two additional clones showed overlapping sequences which span a region of 3065 nucleotides in size. The 1.5- and 1.8-kb cDNA inserts each contained poly(A) addition signals (1508 and 1761 nucleotides, respectively), terminal poly(A) sequences, and the entire coding region for R-II51 (1204 nucleotides) except for a small number of nucleotides at the 5' end. The 2.2-kb cDNA insert contained 394 nucleotides of the coding region a long 3' untranslated region and two more poly(A) addition signals (3041 and 3059 nucleotides). An amino acid microsequence surrounding the autophosphorylation site of pure rat ovarian R-II51 agreed with the amino acid sequence deduced from the nucleotide sequence of the cDNA. Northern blot analyses demonstrated two major mRNA species (1.8 and 3.2 kb in size) in hormone-primed rat ovaries which were approximately 10- and 50-fold greater than the R-II mRNA content in rat brain and rat heart, respectively. Southern blot analysis of rat liver DNA indicated that a single gene codes for R-II51 mRNA. Structural differences among rat ovarian R-II51, rat heart R-II54, and the known amino acid sequences of bovine R-II and R-I subunits also indicate that the rat ovarian R-II51 subunit is the product of a distinct gene.
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PMID:Molecular cloning, cDNA structure, and regulation of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovarian granulosa cells. 242 18

Arginine vasopressin (AVP) potentiates corticotrope responses to CRH by increasing the percentage of target cells that secrete in a reverse hemolytic plaque assay for ACTH. The present studies were designed to test more specific effects of AVP and its second messengers on CRH binding to individual corticotropes. Spectrophotometric analyses of 560 corticotropes from fractions enriched to 90% by counterflow centrifugation showed a 30% increase in the average area of the dark blue label for biotinylated CRH after a 1-h exposure to 10 nM AVP or after activation of protein kinase-C [by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or L calcium channels (by Bay K 8644). In addition, computer analysis of the color of the label (wavelength 476-483) showed a 13% increase in saturation (intensity of the blue) and a 23% decrease in brightness (amount of white) after stimulation. The gray level readings of the blue color were also 18% lower after stimulation, which indicates an increase in density (less light transmitted). Taken together, the increases in label area and intensity indicated that activation of L calcium channels or protein kinase-C enhanced CRH binding by individual corticotropes. When mixed pituitary cell populations were analyzed for percentages of labeled cells, exposure to Bay K 8644, TPA, angiotensin II, or AVP resulted in 30-40% increases in the percentage of CRH-bound cells. Dual reactions for biotinylated CRH and ACTH showed that most of the added CRH-bound cells stored ACTH. The effect of exposure to two of the activators was not additive, however. If L calcium channels were blocked with nimodipine, the protein kinase-C-mediated enhancement in CRH binding and ACTH release was blocked, indicating that these actions are dependent on extracellular calcium. In contrast, nimodipine did not block the TPA-mediated enhancement of ACTH storage. These studies show that the potentiation of CRH-mediated ACTH release by AVP or angiotensin II may occur by the enhancement of CRH binding to individual corticotropes. This appears to promote the cytochemical detection of additional CRH-bound corticotropes which may stem from a reserve cell population that normally has levels of CRH receptors or ACTH stores below thresholds needed for detection. The source of these cells (from stem cells or multipotential cells) remains to be determined.
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PMID:Activation of protein kinase C and L calcium channels enhances binding of biotinylated corticotropin-releasing hormone by anterior pituitary corticotropes. 246 51

The effect of the obligatory precursor of prostaglandin biosynthesis, arachidonic acid, on the release of relaxin by porcine luteal cells was examined by use of a reverse hemolytic plaque assay. In this assay, luteal cells were cocultured in monolayers with protein-A-coupled ovine erythrocytes. In the presence of porcine relaxin antiserum and complement, a zone of hemolysis, a plaque, developed around relaxin-releasing luteal cells, identified as large luteal cells (LLCs). The rate of development of plaques in time-course studies and the area of plaques were then used as an index of the rate of relaxin release and cumulative amount of hormone released, respectively. Incubation of collagenase-dispersed luteal cells derived from early pregnant pigs with 0.1-100 microM arachidonic acid (AA) resulted in dose-dependent increases in the rate of plaque formation. Despite AA stimulation, however, only 55-65% of all LLCs formed plaques during the experimental incubation period (up to 12 h). Minimally and maximally effective doses were about 1 and 10 microM, respectively. In the presence of 10 microM AA, maximal plaque formation occurred significantly faster (1-2 h) than in controls (4-8 h; P less than 0.05). The percentage of plaque-forming cells (plaque-forming LLCs) was, likewise, significantly greater in 10 microM AA-treated monolayers than in controls during the first 3-4 h of incubation. Similarly, agents that liberate endogenous AA (phospholipase A2 and melittin) also stimulated relaxin release. The stimulatory effect of AA (10 microM) on relaxin release was almost wholly blocked by a cyclooxygenase inhibitor (ibuprofen; 20 microM); but not by a lipooxygenase inhibitor (nordihydroguaretic acid; 20 microM). However, the same dose of ibuprofen (20 microM) failed to modulate the stimulatory effect of prostaglandin E2 (1 microM) or phorbol diester (4 beta-phorbol 12 beta-myristate 13 alpha-acetate; 50 nM) on the rate of relaxin release. These results indicate that a product(s) of the cyclooxygenase pathway of AA metabolism participates in the control of relaxin release, but that this metabolite(s) is not essential to the biological action of at least one stimulatory secretagogue. Moreover, this metabolite failed to influence a subpopulation of nonresponsive LLCs. These data taken in association with our previous demonstration that the pathways of both calcium mobilization and protein kinase-C activation are implicated in the regulation of relaxin release, are consistent with the view that AA liberation may amplify the actions of other signalling mechanisms.
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PMID:Analysis of relaxin release by cultured porcine luteal cells using a reverse hemolytic plaque assay: effects of arachidonic acid, cyclo- and lipooxygenase blockers, phospholipase A2, and melittin. 250 67

Actin has been measured in subcellular fractions from Rat-1 fibroblasts and in Rous sarcoma virus-transformed Rat-1 cells (VIT), using the DNase 1 inhibition assay. The transformed cells showed a significant shift in the actin monomer (G)in equilibrium with polymer (F) equilibrium within the cell cytosol, and a significant increase in actin in the Triton-insoluble cytoskeletal core in comparison with untransformed cells. This incorporation of actin into the cytoskeletal core fraction is associated with a change in filamentous actin assemblies from 'stress fibre' patterns to punctate filament aggregates. These differences have been correlated with changes in morphology, in actin, vinculin and alpha-actinin distribution, in adhesion plaque formation and with the production of pp60v-src-associated protein kinase activity in the transformed cells. Changes in actin distribution and its polymerization in response to src-gene expression may play an important role in the determination of the transformed cell characteristics.
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PMID:Effect of transformation by Rous sarcoma virus on the character and distribution of actin in Rat-1 fibroblasts: a biochemical and microscopical study. 301 Oct 50

The primary structure of the beta chain of human glycoprotein Ib (GPIb), the platelet receptor for von Willebrand factor, has been established by a combination of cDNA cloning and amino acid sequence analysis. A lambda phage cDNA expression library prepared from human erythroleukemia cells (HEL cells) was screened with a radiolabeled affinity-purified rabbit polyclonal antibody to the beta chain of GPIb. Eighteen positive clones were isolated and plaque-purified and the nucleotide sequences of three were determined. The composite sequence spanned 968 nucleotides and included a 5' untranslated region of 22 nucleotides, an open reading frame of 618 nucleotides encoding a signal peptide of 28 amino acids and a mature protein of 181 amino acids, a stop codon, and a 3' noncoding region of 307 nucleotides. The 3' noncoding sequence also contained a polyadenylylation signal (AATAAA) 14 nucleotides upstream from the poly(A) tail of 18 nucleotides. Edman degradation of the intact beta chain and of peptides produced by chemical cleavage yielded amino acid sequences spanning 76 residues that were identical to those predicted from the cDNA. The amino-terminal region of the beta chain contains a leucine-rich sequence of 24 amino acids that is similar to a sequence that occurs as seven tandem repeats in the alpha chain of GPIb and nine tandem repeats in leucine-rich alpha 2-glycoprotein. The leucine-rich sequence in the beta chain of GPIb is flanked on both sides by amino acid sequences that are similar to those flanking the leucine-rich tandem repeats of the alpha chain of GPIb and leucine-rich alpha 2-glycoprotein. The amino-terminal region of the beta chain of GPIb is followed by a transmembrane segment of 25 amino acids and an intracellular segment of 34 amino acids at the carboxyl terminus of the protein. The intracellular segment contains an unpaired cysteine and two potential sites for phosphorylation by cAMP-dependent protein kinase.
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PMID:The alpha and beta chains of human platelet glycoprotein Ib are both transmembrane proteins containing a leucine-rich amino acid sequence. 335 70

A purine analogue, 2-aminopurine, reported to act as an inhibitor of protein kinase, selectively, reversibly and in a dose-dependent manner blocked a very early stage in interferon induction. With chick embryo cells and mouse L cells as hosts, and different viral inducers of interferon, maximal effects of 2-aminopurine were observed during the first 4 h of induction. At 10 mM-2-aminopurine there was a 20-fold reduction in the yield of interferon from both cell types. 2-Aminopurine and actinomycin D both prevented interferon induction with the same time course, indicating a transcriptional block to induction; however, only the action of the former was reversed upon removal of the drug. Addition of 2-aminopurine to an agarose overlay resulted in high efficiency plaque formation by vesicular stomatitis virus New Jersey (Hazelhurst) under conditions where endogenous induction of interferon and its feedback action on aged chick embryo cells normally prevented plaque formation. Two other inducible systems, representing genes involved in interferon action (both its development and activation), and those of heat shock, were not affected by 2-aminopurine. A model is presented implicating the interferon-inducible dsRNA-dependent protein kinase as an interferon induction receptor which, on interaction with dsRNA, generates an amplified signal via phosphorylation that ultimately derepresses the interferon gene(s).
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PMID:Interferon induction by viruses. XVI. 2-Aminopurine blocks selectively and reversibly an early stage in interferon induction. 339 21


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