Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 7 de 7
Filter
Add more filters










Database
Language
Publication year range
1.
J Biol Chem ; 276(45): 42580-7, 2001 Nov 09.
Article in English | MEDLINE | ID: mdl-11546803

ABSTRACT

Treatment of macrophages with pyridinyl imidazole inhibitors of p38 protein kinases can inhibit lipopolysaccharide-stimulated tumor necrosis factor alpha secretion. However, bone marrow-derived macrophages from tristetraprolin (TTP)-deficient mice were less sensitive than normal macrophages to this effect of p38 inhibitors, despite evidence for normal p38 activation in response to lipopolysaccharide. TTP is known to cause decreased stability of tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor mRNAs after binding to an AU-rich element in their 3'-untranslated regions. A recombinant TTP fusion protein could be phosphorylated by a recombinant p38 kinase in cell-free assays and was phosphorylated to the same extent by immunoprecipitated p38 derived from normal and TTP-deficient cells stimulated with lipopolysaccharide; in both cases, the enzyme activity was inhibited by the p38 inhibitors. TTP phosphorylation also was increased in intact macrophages after lipopolysaccharide stimulation, an effect that was blocked by the p38 inhibitors. Finally, TTP in mammalian cell extracts bound less well to an AU-rich element RNA probe than did the same amount of TTP following dephosphorylation. These results suggest that TTP may be a component of the signaling cascade, initiated by inflammatory stimuli and mediated in part by activation of p38, that ultimately leads to enhanced secretion of tumor necrosis factor alpha.


Subject(s)
DNA-Binding Proteins , Enzyme Inhibitors/pharmacology , Immediate-Early Proteins/physiology , Mitogen-Activated Protein Kinases/physiology , 3' Untranslated Regions/metabolism , Animals , Interleukin-3/genetics , Macrophages/metabolism , Mice , Mice, Knockout , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Phosphorylation , RNA, Messenger/analysis , Recombinant Fusion Proteins/metabolism , Tristetraprolin , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/genetics , p38 Mitogen-Activated Protein Kinases
2.
Gene ; 267(1): 71-87, 2001 Apr 04.
Article in English | MEDLINE | ID: mdl-11311557

ABSTRACT

The sequencing of expressed sequence tags (ESTs) from Xenopus laevis has lagged behind efforts on many other common experimental organisms and man, partly because of the pseudotetraploid nature of the Xenopus genome. Nonetheless, large collections of Xenopus ESTs would be useful in gene discovery, oligonucleotide-based knockout studies, gene chip analyses of normal and perturbed development, mapping studies in the related diploid frog X. tropicalis, and for other reasons. We have created a normalized library of cDNAs from unfertilized Xenopus eggs. These cells contain all of the information necessary for the first several cell divisions in the early embryo, as well as much of the information needed for embryonic pattern formation and cell fate determination. To date, we have successfully sequenced 13,879 ESTs out of 16,607 attempts (83.6% success rate), with an average sequence read length of 508 bp. Using a fragment assembly program, these ESTs were assembled into 8,985 'contigs' comprised of up to 11 ESTs each. When these contigs were used to search publicly available databases, 46.2% bore no relationship to protein or DNA sequences in the database at the significance level of 1e-6. Examination of a sample of 100 of the assembled contigs revealed that most ( approximately 87%) were comprised of two apparent allelic variants. Expression profiles of 16 of the most prominent contigs showed that 12 exhibited some degree of zygotic expression. These findings have implications for sequence-specific applications for Xenopus ESTs, particularly the use of allele-specific oligonucleotides for knockout studies, differential hybridization techniques such as gene chip analysis, and the establishment of accurate nomenclature and databases for this species.


Subject(s)
Environmental Health , Expressed Sequence Tags , National Institutes of Health (U.S.) , Xenopus/genetics , Alleles , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary/chemistry , DNA, Complementary/genetics , Databases, Factual , Female , Gene Expression Profiling , Gene Expression Regulation , Gene Frequency , Gene Library , Genetic Variation , Molecular Sequence Data , Ovum/metabolism , RNA, Messenger/genetics , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Species Specificity , United States
3.
J Biol Chem ; 275(23): 17827-37, 2000 Jun 09.
Article in English | MEDLINE | ID: mdl-10751406

ABSTRACT

Macrophages derived from tristetraprolin (TTP)-deficient mice exhibited increased tumor necrosis factor alpha (TNFalpha) release as a consequence of increased stability of TNFalpha mRNA. TTP was then shown to destabilize TNFalpha mRNA after binding directly to the AU-rich region (ARE) of the 3'-untranslated region of the TNFalpha mRNA. In mammals and in Xenopus, TTP is the prototype of a small family of three known zinc finger proteins containing two CCCH zinc fingers spaced 18 amino acids apart; a fourth more distantly related family member has been identified in Xenopus and fish. We show here that representatives of all four family members were able to bind to the TNFalpha ARE in a cell-free system and, in most cases, promote the breakdown of TNFalpha mRNA in intact cells. Because the primary sequences of these CCCH proteins are most closely related in their tandem zinc finger domains, we tested whether various fragments of TTP that contained both zinc fingers resembled the intact protein in these assays. We found that amino- and carboxyl-terminal truncated forms of TTP, as well as a 77 amino acid fragment that contained both zinc fingers, could bind to the TNFalpha ARE in cell-free cross-linking and gel shift assays. In addition, these truncated forms of TTP could also stimulate the apparent deadenylation and/or breakdown of TNFalpha mRNA in intact cells. Alignments of the tandem zinc finger domains from all four groups of homologous proteins have identified invariant residues as well as group-specific signature amino acids that presumably contribute to ARE binding and protein-specific activities, respectively.


Subject(s)
3' Untranslated Regions/metabolism , DNA-Binding Proteins , Immediate-Early Proteins , Membrane Proteins/metabolism , Peptide Termination Factors/metabolism , Proteins/metabolism , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Saccharomyces cerevisiae Proteins , Xenopus Proteins , Zinc Fingers , 3' Untranslated Regions/chemistry , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Evolution, Molecular , Humans , Membrane Proteins/chemistry , Mice , Molecular Sequence Data , Peptide Termination Factors/chemistry , Phylogeny , Proteins/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Transfection , Tristetraprolin , Xenopus
4.
Mol Cell Biol ; 19(6): 4311-23, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10330172

ABSTRACT

Mice deficient in tristetraprolin (TTP), the prototype of a family of CCCH zinc finger proteins, develop an inflammatory syndrome mediated by excess tumor necrosis factor alpha (TNF-alpha). Macrophages derived from these mice oversecrete TNF-alpha, by a mechanism that involves stabilization of TNF-alpha mRNA, and TTP can bind directly to the AU-rich element (ARE) in TNF-alpha mRNA (E. Carballo, W. S. Lai, and P. J. Blackshear, Science 281:1001-1005, 1998). We show here that TTP binding to the TNF-alpha ARE is dependent upon the integrity of both zinc fingers, since mutation of a single cysteine residue in either zinc finger to arginine severely attenuated the binding of TTP to the TNF-alpha ARE. In intact cells, TTP at low expression levels promoted a decrease in size of the TNF-alpha mRNA as well as a decrease in its amount; at higher expression levels, the shift to a smaller TNF-alpha mRNA size persisted, while the accumulation of this smaller species increased. RNase H experiments indicated that the shift to a smaller size was due to TTP-promoted deadenylation of TNF-alpha mRNA. This CCCH protein is likely to be important in the deadenylation and degradation of TNF-alpha mRNA and perhaps other ARE-containing mRNAs, both in normal physiology and in certain pathological conditions.


Subject(s)
DNA-Binding Proteins , Immediate-Early Proteins , Proteins/pharmacology , Tumor Necrosis Factor-alpha/drug effects , Base Sequence , Blotting, Northern , Blotting, Western , Cell Line , Cytosol/metabolism , Dactinomycin/pharmacology , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Green Fluorescent Proteins , Kinetics , Luminescent Proteins/metabolism , Macrophages/metabolism , Molecular Sequence Data , Plasmids , Precipitin Tests , Protein Processing, Post-Translational , Protein Synthesis Inhibitors/pharmacology , Proteins/metabolism , RNA, Messenger , Recombinant Fusion Proteins , Ribonuclease H/metabolism , Tissue Distribution , Tristetraprolin , Zinc Fingers
5.
J Biol Chem ; 272(46): 29290-300, 1997 Nov 14.
Article in English | MEDLINE | ID: mdl-9361009

ABSTRACT

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a high affinity cellular substrate for protein kinase C. The MARCKS gene is under multiple modes of transcriptional control, including cytokine- and transformation-dependent, cell-specific, and developmental regulation. This study evaluated the transcriptional control of MARCKS gene expression during early development of Xenopus laevis. Xenopus MARCKS was highly conserved with its mammalian and avian homologues; its mRNA and protein were abundant in the maternal pool and increased after the mid-blastula transition (MBT). The Xenopus MARCKS gene was similar to those of other species, except that a second intron interrupted the 5'- untranslated region. By transiently transfecting XTC-2 cells and microinjecting Xenopus embryos with reporter gene constructs containing serial deletions of 5'-flanking MARCKS sequences, we identified a 124-base pair minimal promoter that was critical for promoter activity. Developmental gel shift assays revealed that a CBF/NF-Y/CP-1-like factor and an Sp1-like factor bound to this region in a manner correlating with the onset of Xenopus MARCKS transcription at MBT. Mutations in the promoter that abolished binding of these two factors also completely inhibited transcriptional activation of the MARCKS gene at MBT. The binding sites for these two factors are highly conserved in the human and mouse MARCKS promoters, suggesting that these elements might also regulate MARCKS transcription in other species. These studies not only increase our knowledge of the transcriptional regulation of the MARCKS genes but also have implications for the mechanisms responsible for zygotic activation of the Xenopus genome at MBT.


Subject(s)
Gene Expression Regulation, Developmental , Intracellular Signaling Peptides and Proteins , Membrane Proteins , Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cell Line , Cell Nucleus/metabolism , Cloning, Molecular , DNA, Complementary , Humans , Mice , Molecular Sequence Data , Myristoylated Alanine-Rich C Kinase Substrate , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid , Sequence Homology, Amino Acid , Sp1 Transcription Factor/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Xenopus laevis/embryology
6.
J Biol Chem ; 262(25): 12356-64, 1987 Sep 05.
Article in English | MEDLINE | ID: mdl-3624263

ABSTRACT

Stimulation of epidermal growth factor (EGF) receptor autophosphorylation by EGF and phosphorylation of a Mr 52,000 protein endogenous to the membrane extracts were decreased 6-12-fold in liver membrane extracts from mice homozygous for either the ob/ob or db/db mutation when compared to controls. Liver membranes from the mutant mice bound 4-5-fold less 125I-EGF/unit of protein than did their normal littermates, but exhibited normal EGF binding affinity. Similar decreases in EGF binding were noted in liver membranes from homozygous fa/fa Zucker rats, another obese, hyperinsulinemic animal model, when compared to values from control animals. We also immunoprecipitated hepatic EGF receptors from mice injected with [35S]methionine, and found that livers from db/db mice contained approximately 35% of the labeled EGF receptors found in control animals. Both ob/ob and db/db mice had serum immunoreactive EGF levels similar to or lower than those found in unaffected littermates, suggesting that ligand-mediated down-regulation of receptors was not the cause of the decreased EGF binding. In one mutant, db/db, the decreased binding was associated with a 6-fold decrease in the levels of liver EGF receptor mRNA transcripts; in the ob/ob mice, at most a 2-fold decrease in the level of liver EGF receptor transcripts was observed. EGF binding to cultured peritoneal fibroblasts derived from db/db mice was normal, suggesting that the abnormality in the mutant mice might result from altered environmental or tissue-specific factors rather than an abnormal receptor gene. This was supported by Southern blot analysis of DNA from these animals, which showed identical restriction fragment patterns for the EGF receptor gene in both control and mutant animals. These data indicate that three distinct strains of obese hyperglycemic rodents have decreased levels of hepatic EGF receptors, and suggest that this decrease may result from altered environmental stimuli or tissue-specific factors rather than a primary defect in the EGF receptor gene.


Subject(s)
ErbB Receptors/metabolism , Hyperglycemia/metabolism , Liver/metabolism , Obesity/metabolism , Animals , ErbB Receptors/genetics , Hyperglycemia/genetics , Methionine/metabolism , Mice , Mice, Inbred C57BL , Mice, Obese , Molecular Weight , Obesity/genetics , RNA, Messenger/analysis , Transcription, Genetic
7.
Blood ; 60(2): 333-9, 1982 Aug.
Article in English | MEDLINE | ID: mdl-6953983

ABSTRACT

Phorbol esters are potent stimulants of the respiratory burst of the human neutrophil as assessed by superoxide (O2-) generation in whole cells and by NADPH-oxidase activity in a broken-cell 27,000-g particulate fraction. Phorbol 12-myristate, 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) stimulate production of O2- by human neutrophils with ED50 concentrations of 3.9 +/- 2.1 and 41.7 +/- 7.1 nM, respectively. The relation of biologic activity to receptor occupancy was assessed with binding studies of PMA and PDBu. Phorbol ester binding revealed a single high affinity phorbol ester receptor present at 7.6 x 10(5) sites/cell. The binding affinities for PMA and PDBu, 4.9 nM and 38.4 nM, respectively, agreed quantitatively with that of biologic potencies. Because of the high concentration of phorbol ester receptors (up to 125 nM) and the large amount of nonspecific binding at high cell density, apparent discrepancies between ED50's for NADPH-oxidase and whole cell O2- generation were noted. With the use of low cell concentrations, quantitative agreement between intact cell production of O2-, NADPH-oxidase activity, and receptor binding was found. These results further support the identity of the NADPH-oxidase as the enzymatic source of respiratory burst O2- production in human neutrophils.


Subject(s)
Caenorhabditis elegans Proteins , NADH, NADPH Oxidoreductases/metabolism , Neutrophils/metabolism , Protein Kinase C , Receptors, Drug/metabolism , Carrier Proteins , Humans , Neuraminidase/metabolism , Peroxides/biosynthesis , Phorbol 12,13-Dibutyrate , Phorbol Esters/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...