Browsing by keyword "Heat"

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    • Mechanism of sodium arsenite-mediated induction of heme oxygenase-1 in hepatoma cells. Role of mitogen-activated protein kinases
      Elbirt, Kimberly K.; Whitmarsh, Alan J.; Davis, Roger J.; Bonkovsky, Herbert L. (1998-05-16)
      Heme oxygenase-1 is an inducible enzyme that catalyzes heme degradation and has been proposed to play a role in protecting cells against oxidative stress-related injury. We investigated the induction of heme oxygenase-1 by the tumor promoter arsenite in a chicken hepatoma cell line, LMH. We identified a heme oxygenase-1 promoter-driven luciferase reporter construct that was highly and reproducibly expressed in response to sodium arsenite treatment. This construct was used to investigate the role of mitogen-activated protein (MAP) kinases in arsenite-mediated heme oxygenase-1 gene expression. In LMH cells, sodium arsenite, cadmium, and heat shock, but not heme, induced activity of the MAP kinases extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. To examine whether these MAP kinases were involved in mediating heme oxygenase-1 gene expression, we utilized constitutively activated and dominant negative components of the ERK, JNK, and p38 MAP kinase signaling pathways. Involvement of an AP-1 site in arsenite induction of heme oxygenase-1 gene expression was studied. We conclude that the MAP kinases ERK and p38 are involved in the induction of heme oxygenase-1, and that at least one AP-1 element (located -1576 base pairs upstream of the transcription start site) is involved in this response.

    • Pertussis toxin blocks the inhibitory effects of calcitonin on cyclic AMP accumulation in stimulated cultured human monocytes
      Stock, John L.; Coderre, James A. (1987-11-01)
      Surface stimulation of fresh or cultured human mononuclear cells by latex particles causes an increase in the accumulation of cyclic AMP that is inhibited by preincubation with calcitonin (CT). Preincubation of cultured monocytes with 500 ng/ml pertussis toxin totally blocks the inhibitory effects of CT at low concentrations of this hormone. The effects of pertussis toxin are dose-related and eliminated by boiling the toxin. Similar preincubations with cholera toxin have no significant effects on subsequent inhibition of surface-stimulated cyclic AMP by CT. Membranes prepared from cultured human monocytes contain a 41,000-dalton protein that is ADP-ribosylated by pertussis toxin and may be the inhibitory guanine nucleotide regulatory protein (Ni) mediating this inhibition.

    • Purification of calmodulin from Chlamydomonas: calmodulin occurs in cell bodies and flagella
      Gitelman, Stephen E.; Witman, George B. (1980-12-01)
      Calmodulin has been purified from cell bodies of the green alga Chlamydomonas by Ca++-dependent affinity chromatography on fluphenazine-Sepharose 4B. Calmodulin from this primitive organism closely resembles that from bovine brain in a number of properties, including (a) binding to fluphenazine in a Ca++-dependent, reversible manner, (b) functioning as a heat-stable, Ca++-dependent activator of cyclic nucleotide phosphodiesterase, and (c) electrophoretic mobility in SDS-polyacrylamide gels in both the presence and absence of Ca++, which causes a shift in the relative mobility of calmodulin. Calmodulin has also been identified by the criteria of phosphodiesterase activation and electrophoretic mobility in both the detergent soluble "membrane plus matrix" and the axoneme fractions of Chlamydomonas flagella. Calmodulin is not associated with the partially purified 12S or 18S dynein ATPases of Chlamydomonas. The presence of calmodulin in the flagellum suggests that it is involved in one or more of the Ca++-dependent activities of this organelle.

    • Reconstitution of Golgi vesicle CMP-sialic acid and adenosine 3'-phosphate 5'-phosphosulfate transport into proteoliposomes
      Milla, Marcos E.; Hirschberg, Carlos B. (1989-03-01)
      We have previously shown that Golgi apparatus vesicles transport nucleotide sugars and nucleotide sulfate into their lumen. These transport activities are organelle and substrate specific and are characterized by apparent Km for nucleotide derivatives in the low micromolar range. As part of our goal of purifying and characterizing the above transport proteins, we have reconstituted a protein extract from rat liver Golgi membranes into phosphatidylcholine liposomes. The resulting proteoliposomes transport CMP-N-acetylneuraminic acid (CMP-AcNeu) and adenosine 3'-phosphate 5'-phosphosulfate with very similar affinity and inhibition characteristics as intact Golgi vesicles. Sialic acid and sodium sulfate, which are transported only very slowly into the lumen of Golgi vesicles, are transported at low rates by the reconstituted proteoliposomes. Neither rough endoplasmic reticulum-derived vesicles nor proteoliposomes made from proteins of the rough endoplasmic reticulum transport CMP-AcNeu. The above results demonstrate that this reconstituted system can be used for further purification and characterization of nucleotide sugar and nucleotide sulfate translocator proteins. This approach should also be useful to study membrane transport proteins of lysosomes and endosomes.

    • Regulation of Drosophila p38 activation by specific MAP2 kinase and MAP3 kinase in response to different stimuli
      Zhuang, Zi-Heng; Zhou, Yuan; Yu, Ming-Can; Silverman, Neal S.; Ge, Bao-Xue (2005-07-15)
      The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in cellular responses to inflammatory stimuli and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. However, the mechanism of how different upstream MAP2Ks and MAP3Ks specifically contribute to p38 activation in response to different stimuli is still not clearly understood. By using double-stranded RNA-mediated interference (RNAi) in Drosophila cells, we demonstrate that D-MKK3 is a major MAP2K responsible for D-p38 activation by UV, heat shock, NaCl or peptiodglycan (PGN). Stimulation of UV and PGN activates D-p38 through D-MEKK1, heat shock-induced activation of D-p38 signals through both D-MEKK1 and D-ASK1. On the other hand, maximal activation of D-p38 by NaCl requires the expression of four MAP3Ks.

    • Structure and function of a membrane anchor-less form of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus
      Mirza, Anne M.; Sheehan, John P.; Hardy, Larry W.; Glickman, Rhona L.; Iorio, Ronald M. (1993-10-05)
      The hemagglutinin-neuraminidase (HN) glycoprotein of paramyxoviruses is anchored in the virion membrane near its amino terminus, protruding from the virion surface to mediate attachment to cellular receptors. Solubilization of HN spikes can be achieved by treatment of virions with detergent and high salt concentrations. When the solubilized HN protein from the Australia-Victoria (AV) isolate of the virus is incubated at 37 degrees C, a chymotrypsin-sensitive site between residues 112 and 113 is exposed. A chymotrypsin-cleaved soluble form of the protein, named CT-HN, has been prepared using this approach. It is membrane anchor-less, due to removal of a 14-kDa fragment from the NH2 terminus of HN. It retains all potential glycosylation sites and cysteines present in the ectodomain of the native protein. It migrates in nonreducing gels and sediments in sucrose gradients at the rate expected for homodimeric HN. The latter is also consistent with our demonstration by site-directed mutagenesis that cysteine residues at positions 6 and 123, respectively, mediate disulfide-linked homotetramer and homodimer formation. CT-HN retains almost total antigenicity, suggesting that it is conformationally very similar to the intact molecule, as well as receptor recognition function and, at low pH, neuraminidase activity. It should prove to be a useful tool for further studies of the structure and function of this important viral glycoprotein.

    • The relationship between chain connectivity and domain stability in the equilibrium and kinetic folding mechanisms of dihydrofolate reductase from E.coli
      Svensson, Anna-Karin E.; Zitzewitz, Jill A.; Matthews, C. Robert; Smith, Virginia F. (2006-02-03)
      The role of domains in defining the equilibrium and kinetic folding properties of dihydrofolate reductase (DHFR) from Escherichia coli was probed by examining the thermodynamic and kinetic properties of a set of variants in which the chain connectivity in the discontinuous loop domain (DLD) and the adenosine-binding domain (ABD) was altered by permutation. To test the concept that chain cleavage can selectively destabilize the domain in which the N- and C-termini are resident, permutations were introduced at one position within the ABD, one within the DLD and one at a boundary between the domains. The results demonstrated that a continuous ABD is required for a stable thermal intermediate and a continuous DLD is required for a stable urea intermediate. The permutation at the domain interface had both a thermal and urea intermediate. Strikingly, the observable kinetic folding responses of all three permuted proteins were very similar to the wild-type protein. These results demonstrate a crucial role for stable domains in defining the energy surface for the equilibrium folding reaction of DHFR. If domain connectivity affects the kinetic mechanism, the effects must occur in the sub-millisecond time range.