Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 59
Filter
Add more filters











Publication year range
1.
Dis Aquat Organ ; 84(1): 79-87, 2009 Mar 09.
Article in English | MEDLINE | ID: mdl-19419009

ABSTRACT

Hematodinium sp. is a parasitic dinoflagellate infecting the blue crab Callinectes sapidus and other crustaceans. PCR-based assays are currently being used to identify infections in crabs that would have been undetectable by traditional microscopic examination. We therefore sought to define the limits of quantitative PCR (qPCR) detection within the context of field collection protocols. We present a qPCR assay based on the Hematodinium sp. 18S rRNA gene that can detect 10 copies of the gene per reaction. Analysis of a cell dilution series vs. defined numbers of a cloned Hematodinium sp. 18S rRNA gene suggests a copy number of 10,000 per parasite and predicts a sensitivity of 0.001 cell equivalents. In practice, the assays are based on analysis of 1% of the DNA extracted from 200 microl of serum, yielding a theoretical detection limit of 5 cells ml(-1) hemolymph, assuming that 1 cell is present per sample. When applied to a limited field survey of blue crabs collected in Maryland coastal bays from May to August 2005, 24 of 128 crabs (18.8%) were identified as positive for Hematodinium sp. infection using qPCR. In comparison, only 6 of 128 crabs (4.7%) were identified as positive using traditional hemolymph microscopic examination. The qPCR method also detected the parasite in gill, muscle, heart and hepatopancreas tissues, with 17.2% of the crabs showing infection in at least one of these tissues. Importantly, it is now possible to enumerate parasites within defined quantities of crab tissue, which permits collection of more detailed information on the epizootiology of the pathogen.


Subject(s)
Brachyura/parasitology , Dinoflagellida/isolation & purification , Polymerase Chain Reaction/methods , Animals , Atlantic Ocean , Female , Male , Maryland , Sensitivity and Specificity , Virginia
2.
J Food Prot ; 65(8): 1253-8, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12182476

ABSTRACT

The effect of nisin combined with pulsed electric fields (PEF) and water activity reduction by sodium chloride (NaCl) on the inactivation of E. coli in simulated milk ultrafiltrate media was studied with a Doehlert design and a response surface method. The reduction of water activity from 0.99 to 0.95 by the addition of NaCl (without any other hurdle) did not affect E. coli viability of approximately 10(8) CFU/ml. A reduction in PEF effectiveness occurred when the NaCl concentration was increased because of an increase in conductance, which reduced the pulse decay time. In cells submitted to PEF nisin activity was decreased, probably as a consequence of the nonspecific binding of nisin to cellular debris or the emergence of new binding sites in or from cells. However, the lethal effect due to nisin was reestablished and further improved when water activity was reduced to 0.95. A synergistic effect was evidenced when low-intensity PEF were applied. Decreasing water activity to 0.95 and applying PEF at 5 kV/cm (a nonlethal intensity when no other hurdle is used) with the further addition of nisin (1,200 IU/ml) resulted in a 5-log cycle reduction of the bacterial population.


Subject(s)
Electric Stimulation , Escherichia coli/drug effects , Food Preservation/methods , Nisin/pharmacology , Sodium Chloride/pharmacology , Animals , Anti-Bacterial Agents , Colony Count, Microbial , Escherichia coli/growth & development , Food Microbiology , Milk/microbiology , Pulsatile Flow , Water
4.
J Food Prot ; 64(10): 1510-4, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11601698

ABSTRACT

The Doehlert design and surface response methodology were used to study the influence of pH and water activity (aw) on Escherichia coli inhibition by nisin. Combining stress factors at levels where they are not inhibitory by themselves, a reduction of E. coli survival fraction can be achieved with lower nisin doses than in a single nisin treatment. For all the pH values assayed, a synergistic effect of aw and nisin concentration was detected, and the isoresponse lines showed the existence of an area of maximum inhibition. Factors that reduced viable cell counts by 4 to 5 log cycles were 1,000 to 1,400 IU of nisin per ml at pH 5.5 to 6.5 and a water activity of 0.97 and 0.98. The addition of different ionic and nonionic solutes to control aw suggested that the effect of aw in the inhibitory action of nisin on E. coli cells was not solute-specific. The use of the Doehlert experimental design was effective to determine the optimal combination of stress factors, as well as to point out the most important variables that affected E. coli inhibition.


Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli/drug effects , Nisin/pharmacology , Water/pharmacology , Colony Count, Microbial , Dose-Response Relationship, Drug , Drug Synergism , Escherichia coli/growth & development , Hydrogen-Ion Concentration
5.
Eur J Biochem ; 268(20): 5375-85, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11606200

ABSTRACT

Eukaryotic translation initiation factor 4E (eIF4E) is essential for efficient translation of the vast majority of capped cellular mRNAs; it binds the 5'-methylated guanosine cap of mRNA and serves as a nucleation point for the assembly of the 48S preinitiation complex. eIF4E is phosphorylated in vivo at residue 209 of the human sequence. The phosphorylated form is often regarded as the active state of the protein, with ribosome-associated eIF4E enriched for the phosphorylated form and increased phosphorylation often correlated with upregulation of rates of protein synthesis. However, the only reported measured effect attributable to phosphorylation at the physiological site has been a relatively small increase in the affinity of eIF4E for the mRNA m7GTP cap structure. Here, we provide data to suggest that phosphorylation of eIF4E at Ser209 is not required for translation. eIF4E that is modified such that it cannot be phosphorylated (Ser209-->Ala), is unimpaired in its ability to restore translation to an eIF4E-dependent in vitro translation system. In addition, both the wild-type and mutant forms of eIF4E interact equally well with eIF4G, with the phosphorylation of eIF4E not required to effect the change in conformation of eIF4G that is required for efficient cleavage of eIF4G by L-protease. Furthermore, we show that wild-type and phosphorylation-site variants of eIF4E protein are equally able to rescue the lethal phenotype of eIF4E deletion in S. cerevisiae.


Subject(s)
Peptide Initiation Factors/chemistry , Peptide Initiation Factors/metabolism , Protein Biosynthesis , Serine/metabolism , Animals , Blotting, Western , Cell Extracts , Endopeptidases/metabolism , Eukaryotic Initiation Factor-4E , Eukaryotic Initiation Factor-4G , Humans , Luciferases/genetics , Luciferases/metabolism , Mutation/genetics , Peptide Initiation Factors/genetics , Phosphorylation , Polymerase Chain Reaction , Polyribosomes/chemistry , Polyribosomes/metabolism , Rabbits , Recombinant Proteins/metabolism , Reticulocytes/cytology , Reticulocytes/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development
6.
Curr Protoc Cell Biol ; Chapter 11: Unit 11.2, 2001 May.
Article in English | MEDLINE | ID: mdl-18228307

ABSTRACT

The two most frequently used systems for in vitro translation are the rabbit reticulocyte system and the wheat germ extract. These systems are useful for mRNAs isolated from cells, tissues, and capped or uncapped mRNA produced in vitro by transcription of cDNA. In a combined system, mRNA can be transcribed and translated in a single reaction. In addition these systems can be used for translation reactions with biotinylated amino acids; this allows capture of the newly synthesized protein using streptavidin immobilized on agarose.


Subject(s)
Molecular Biology/methods , Reticulocytes/physiology , Transcription, Genetic/physiology , Animals , Cell-Free System , Female , Humans , In Vitro Techniques , Plant Extracts , RNA, Messenger/physiology , Rabbits , Triticum
7.
Differentiation ; 66(1): 15-22, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10997588

ABSTRACT

Translation initiation factor 4E (eIF4E) binds to the m7GTP cap structure of eukaryotic mRNAs and influences the overall rates of translation. The eIF4E protein is subject to regulation at a number of levels that allow it to modulate translation of maternal mRNAs in early embryos before the onset of zygotic transcription. In zebrafish eIF4E (zeIF4E) mRNA levels are elevated in specific tissues and at specific times during embryogenesis. We have characterized the organization of the zeIF4E gene to facilitate elucidation of the molecular mechanisms that influence its expression. The zeIF4E gene spans about 14 kb and like its human counterpart is comprised of seven exons. Alternative splicing between the first and second exon generates two mRNA splice-forms called SF1 and SF2. Nuclease-S1-protection and primer-extension reveal two zeIF4E transcriptional start-sites. Transcripts initiating from the distal start-site during oogenesis are exclusively SF1, while initiation from the proximal start-site generates both splice-forms. Although translation in vitro of SF1 mRNA gives rise to a protein consistent in mass with affinity-purified zeIF4E, SF2 mRNA does not. Instead, SF2 mRNA inhibits in vitro protein synthesis in a concentration-dependent manner, suggesting it functions as a translational attenuator. Thus, specific transcriptional activation from the distal start-site may provide a unique mechanism for transcriptional regulation of the levels, as well as the function of zeIF4E mRNAs.


Subject(s)
Alternative Splicing/genetics , Peptide Initiation Factors/biosynthesis , Peptide Initiation Factors/genetics , Protein Biosynthesis , Transcription, Genetic/genetics , Zebrafish/genetics , Animals , Base Sequence , Cloning, Molecular , Eukaryotic Initiation Factor-4E , Exons/genetics , Genomic Library , Introns/genetics , Molecular Sequence Data , Nuclease Protection Assays , Peptide Initiation Factors/metabolism , Protein Binding , RNA Caps/genetics , RNA Caps/metabolism , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/biosynthesis , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Zebrafish/metabolism
8.
J Food Prot ; 63(6): 741-6, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10852567

ABSTRACT

The Doehlert design was applied in order to investigate the combined effect of nisin and high voltage pulsed electric fields (PEF) on the inactivation of Escherichia coli in simulated milk ultrafiltrate media. Nisin alone was totally inactivated by PEF, but in the presence of bacterial cells a protective effect was observed. However, the effectiveness of nisin was still decreased when bacterial cells were subjected to the combined treatment. In spite of this phenomenon, an almost additive response emerged as a consequence of the combined treatment. A 4-log cycle reduction may be accomplished with around 1,000 IU/ml (7.15 microM) of nisin and three pulses of 11.25 kV/cm or 500 IU/ml for five pulses of the same intensity. The observed efficacy arising from the combination of both treatments suggests the possibility of using PEF for improving the action spectrum of natural antimicrobials.


Subject(s)
Anti-Bacterial Agents/pharmacology , Electric Stimulation , Escherichia coli , Food Preservation/methods , Nisin/pharmacology , Animals , Escherichia coli/drug effects , Filtration , Milk/microbiology , Pulsatile Flow
9.
Virology ; 257(2): 303-13, 1999 May 10.
Article in English | MEDLINE | ID: mdl-10329541

ABSTRACT

The 2'-5' oligoadenylate synthetases and the protein kinase PKR are both interferon-induced, double-stranded RNA-dependent proteins that play important roles in the antiviral effects of the interferons and in cellular growth control. Both enzymes are activated by natural or synthetic dsRNAs and by single-stranded RNAs that possess extensive secondary structure. This report describes the effects of the small Epstein-Barr virus-encoded RNA EBER-1 on the regulation of 2-5(A) synthetase activity. We demonstrate that EBER-1 RNA binds to and activates the human 40-kDa 2-5(A) synthetase in a dose-dependent manner. The efficiency of EBER-1 as an activator of 2-5(A) synthetase is approximately 25% of that of the synthetic double-stranded RNA poly(I)/poly(C), and poly(I)/poly(C) further stimulates enzyme activity even in the presence of a high concentration of EBER-1. Conversely, EBER-1 neither stimulates nor inhibits 2-5(A) synthetase that has been activated by a high concentration of poly(I)/poly(C). Competitive binding assays suggest that the relative affinity of the enzyme for poly(I)/poly(C) is considerably higher than that for EBER-1. Our data indicate that EBER-1, like VAI RNA of adenovirus, TAR RNA of HIV-1, and Rex-RE RNA of HTLV-1, is able to activate the 2-5(A) synthetases. The significance of why several viruses may activate the 2-5(A) synthetase/RNase L-mediated RNA degradation pathway is discussed.


Subject(s)
2',5'-Oligoadenylate Synthetase/metabolism , Herpesvirus 4, Human/genetics , RNA, Viral/metabolism , 2',5'-Oligoadenylate Synthetase/genetics , Animals , Cell Line , Enzyme Activation , Humans , Interferon Inducers/metabolism , Interferons , Poly I-C/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Spodoptera/cytology
10.
Int J Biochem Cell Biol ; 31(1): 123-38, 1999 Jan.
Article in English | MEDLINE | ID: mdl-10216948

ABSTRACT

The double-stranded (ds) RNA-regulated serine/threonine protein kinase, PKR, is an interferon-inducible enzyme of widespread occurrence in mammalian cells. PKR is activated by dsRNA via a mechanism involving autophosphorylation. Once activated, the enzyme phosphorylates the alpha-subunit of protein synthesis initiation factor eIF2, thereby inhibiting translation. Accumulating data suggest that PKR has additional substrates, and that the kinase may also regulate gene transcription and signal transduction pathways. Although PKR plays an important role in mediating the antiviral effects of interferons, PKR is also implicated in regulating cell proliferation in uninfected cells and may have a tumor suppressor function under normal conditions. Studies of human malignancies and tumor cell lines suggest that, in general, patients bearing tumors with a higher PKR content have a more favorable prognosis. However, in human breast carcinoma cells, dysregulation of PKR may be associated with the establishment or maintenance of the transformed state.


Subject(s)
Apoptosis/physiology , Neoplasms/enzymology , Proto-Oncogene Proteins c-bcl-2 , RNA-Binding Proteins/metabolism , Repressor Proteins/metabolism , eIF-2 Kinase/metabolism , Animals , Base Sequence , Breast Neoplasms/enzymology , Carcinoma/enzymology , Cell Differentiation/physiology , Cell Division , Cell Transformation, Neoplastic , Humans , Molecular Sequence Data , Poly(A)-Binding Proteins , Protein Biosynthesis , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA-Binding Proteins/genetics , Repressor Proteins/chemistry , Repressor Proteins/genetics , bcl-2-Associated X Protein , eIF-2 Kinase/chemistry , eIF-2 Kinase/genetics
11.
Int J Biochem Cell Biol ; 31(1): 175-89, 1999 Jan.
Article in English | MEDLINE | ID: mdl-10216952

ABSTRACT

The interferon induced, dsRNA-activated, protein kinase, PKR, is a key regulator of translational initiation, playing an important role in the regulation of cell proliferation, apoptosis and transformation. PKR levels correlate inversely with proliferative activity in several human tumor systems. This inverse relationship breaks down in human invasive ductal breast carcinomas which exhibit high levels of PKR (Haines et al., Tumor Biol. 17 (1996) 5-12). Consistent with the data from human tumors, the levels of PKR in several breast carcinoma cell lines, MCF7, T47D, BT20, MDAMB231 and MDAMB468, are paradoxically high compared to those found in the normal breast cell lines MCF10A and Hs578Bst. The activity of affinity- or immuno-purified PKR from MCF7, T47D, and BT20 cells appears to be severely attenuated, as judged by its ability to autophosphorylate, or phosphorylate eIF2 alpha. Furthermore, the activity of the kinase from breast carcinoma cells is refractory to stimulation by dsRNA or heparin. However, PKR from breast carcinoma cells remains functional with respect to its ability to bind dsRNA. The activity of PKR from MCF10A cells is reduced by prior incubation with extracts from MCF7 cells, suggesting that MCF7 extracts contain a transdominant inhibitor of PKR. Deregulation of PKR may therefore provide a mechanism for the development or maintenance of a transformed phenotype of human breast carcinomas, mimicking the effects of manipulation of PKR or eIF2 activity observed in experimental systems. Thus, breast carcinomas may provide the first indication of a role for PKR in the pathogenesis of a naturally occurring human cancer.


Subject(s)
Breast Neoplasms/enzymology , eIF-2 Kinase/metabolism , Breast/enzymology , Breast Neoplasms/drug therapy , Cell Extracts/pharmacology , Cytoplasm/chemistry , Enzyme Activation/drug effects , Eukaryotic Initiation Factor-2/metabolism , Female , HeLa Cells/drug effects , HeLa Cells/enzymology , Heparin/pharmacology , Humans , Interferon-beta/pharmacology , Phosphorylation , Poly C/pharmacology , Poly I/pharmacology , Tumor Cells, Cultured , eIF-2 Kinase/drug effects
12.
Mol Cell Biol ; 18(11): 6493-504, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9774665

ABSTRACT

Interferons (IFNs) and retinoids are potent biological response modifiers. By using JAK-STAT pathways, IFNs regulate the expression of genes involved in antiviral, antitumor, and immunomodulatory actions. Retinoids exert their cell growth-regulatory effects via nuclear receptors, which also function as transcription factors. Although these ligands act through distinct mechanisms, several studies have shown that the combination of IFNs and retinoids synergistically inhibits cell growth. We have previously reported that IFN-beta-all-trans-retinoic acid (RA) combination is a more potent growth suppressor of human tumor xenografts in vivo than either agent alone. Furthermore, the IFN-RA combination causes cell death in several tumor cell lines in vitro. However, the molecular basis for these growth-suppressive actions is unknown. It has been suggested that certain gene products, which mediate the antiviral actions of IFNs, are also responsible for the antitumor actions of the IFN-RA combination. However, we did not find a correlation between their activities and cell death. Therefore, we have used an antisense knockout approach to directly identify the gene products that mediate cell death and have isolated several genes associated with retinoid-IFN-induced mortality (GRIM). In this investigation, we characterized one of the GRIM cDNAs, GRIM-12. Sequence analysis suggests that the GRIM-12 product is identical to human thioredoxin reductase (TR). TR is posttranscriptionally induced by the IFN-RA combination in human breast carcinoma cells. Overexpression of GRIM-12 causes a small amount of cell death and further enhances the susceptibility of cells to IFN-RA-induced death. Dominant negative inhibitors directed against TR inhibit its cell death-inducing functions. Interference with TR enzymatic activity led to growth promotion in the presence of the IFN-RA combination. Thus, these studies identify a novel function for TR in cell growth regulation.


Subject(s)
Apoptosis/drug effects , Interferons/pharmacology , Thioredoxin-Disulfide Reductase/physiology , Tretinoin/pharmacology , Amino Acid Sequence , Breast Neoplasms/enzymology , Cell Cycle/drug effects , Cell Division/drug effects , Flow Cytometry , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Humans , Molecular Sequence Data , Neoplasm Proteins/chemistry , Oligonucleotides, Antisense/pharmacology , Sequence Analysis , Tumor Cells, Cultured
13.
Virology ; 250(2): 302-15, 1998 Oct 25.
Article in English | MEDLINE | ID: mdl-9792841

ABSTRACT

The vaccinia virus E3L gene product, pE3, is a dsRNA binding protein that prevents activation of the interferon-induced, dsRNA-activated protein kinase, PKR. Activation of PKR, which results in phosphorylation of the translation initiation factor, eIF2alpha, leads to the inhibition of protein synthesis, a process involved in defense against virus infection. The E3L gene product has a conserved dsRNA binding domain (DRBD) in its carboxyl-terminal region and has been shown to function in vitro by sequestration of dsRNA. We have utilized in vitro binding assays and the yeast two-hybrid system to demonstrate direct interactions of pE3 with PKR. By these methods, we demonstrate that pE3 interacts with two distinct regions in PKR, the amino-terminal (amino acids 1-99) located in the regulatory domain and the carboxyl-terminal (amino acids 367-523) located in the catalytic domain. The amino-terminal region of PKR that interacts with pE3 contains a conserved DRBD, suggesting that PKR can form nonfunctional heterodimers with pE3, analogous to those seen with other dsRNA binding proteins. Interaction of pE3 with the amino-terminal region of PKR is enhanced by dsRNA. In contrast, dsRNA reduces the interaction of pE3 with the carboxyl-terminal region of PKR. Competition experiments demonstrate that the carboxyl-terminal region of PKR, to which pE3 binds, overlaps the region with which eIF2alpha and the pseudosubstrate pK3 interact, suggesting that pE3 may also prevent PKR activation by masking the substrate binding domain. Like pE3, the amino-terminal region of PKR also interacts with the carboxyl-terminal domain of PKR. These interactions increase our understanding of the mechanisms by which pE3 downregulates PKR. In addition, the PKR-PKR interactions observed leads us to suggest a novel autoregulatory mechanism for activation of PKR in which dsRNA binding to the DRBD(s) induces a conformational change that results in release of the amino terminal region from the substrate binding domain, allowing access to eIF2alpha and its subsequent phosphorylation.


Subject(s)
RNA-Binding Proteins/metabolism , Vaccinia virus/metabolism , Viral Proteins/metabolism , eIF-2 Kinase/metabolism , Binding Sites , Binding, Competitive , Catalysis , HeLa Cells , Humans , Nucleic Acid Hybridization , RNA, Double-Stranded/metabolism , Saccharomyces cerevisiae , Sulfur Radioisotopes
14.
Protein Expr Purif ; 12(3): 415-9, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9535710

ABSTRACT

The alpha-subunit of eukaryotic initiation factor eIF2 (eIF2alpha) plays an important role in the regulation of mRNA translation through modulation of the interaction of eIF2 and a second initiation factor, eIF2B. The interaction of the two proteins is regulated in vivo by phosphorylation of eIF2alpha at Ser51. In the present study, rat eIF2alpha was expressed in Sf21 cells using the baculovirus expression system. The recombinant protein was purified to >90% homogeneity in a single immunoaffinity chromatographic step. The protein was free of endogenous eIF2alpha kinase activity and was rapidly phosphorylated by the eIF2alpha kinases HCR and PKR. A variant of eIF2alpha in which the phosphorylation site was changed to Ala was also expressed and purified. The variant eIF2alpha was not phosphorylated by either HCR or PKR, demonstrating that the kinases specifically phosphorylate the correct site in the recombinant protein even in the absence of the other two subunits of the protein. In summary, a rapid and inexpensive method for obtaining eIF2alpha has been developed. Use of the wildtype and variant forms of eIF2alpha to measure eIF2alpha kinase activity in cell and tissue extracts should greatly facilitate examination of the regulation of mRNA translation under a variety of conditions.


Subject(s)
Eukaryotic Initiation Factor-2/isolation & purification , Gene Expression Regulation/genetics , Animals , Baculoviridae/physiology , Base Sequence , Cell Line , Chromatography, Affinity , DNA Primers/chemistry , Electrophoresis, Polyacrylamide Gel , Eukaryotic Initiation Factor-2/genetics , Eukaryotic Initiation Factor-2/metabolism , Heme/pharmacology , Phosphorylation , Polymerase Chain Reaction , Rats , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Repressor Proteins/drug effects , Repressor Proteins/metabolism , Spodoptera/cytology , Spodoptera/virology , Time Factors , eIF-2 Kinase/metabolism
15.
Methods ; 11(4): 419-25, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9126555

ABSTRACT

The combination of vertical, one-dimensional isoelectric focusing and immunoblotting works very well for the evaluation of the phosphorylation state of the alpha-subunit of eIF2 using reticulocyte lysate or purified eIF2. However, the method is more difficult to apply to the analysis of eIF2 alpha phosphorylation in cultured cells. In part this reflects the fact that the protein content of cultured cell extracts is rarely as high as that found in extracts produced from reticulocytes, and in part this reflects the fact that some component(s) of cell extracts interferes with the entry of eIF2 alpha into the isoelectric focusing gel. To overcome these difficulties, we have modified the earlier method to include immunoprecipitation of eIF2 from cell extracts prior to isoelectric focusing, as well as a low sodium dodecyl sulfate concentration in the isoelectric focusing sample buffer. Since the PKR activation state and therefore the eIF2 alpha phosphorylation state change with cell density and nutritional status, we routinely set up consistent feeding schedules and recommend the collection of data over a range of cell densities.


Subject(s)
Eukaryotic Initiation Factor-2/metabolism , Isoelectric Focusing/methods , 3T3 Cells , Animals , Blotting, Western , Cells, Cultured , Evaluation Studies as Topic , Mice , Phosphorylation , Precipitin Tests
16.
Eur J Biochem ; 250(1): 85-91, 1997 Nov 15.
Article in English | MEDLINE | ID: mdl-9431994

ABSTRACT

The vaccinia virus K3L gene product, pK3, binds to the dsRNA-activated protein kinase, PKR, reducing its ability to interact with and phosphorylate eIF2alpha. On the basis of this characteristic and the homology of pK3 to the N-terminus of eIF2alpha, several laboratories have utilized pK3 to investigate the molecular determinants that specify substrate recognition by PKR. The data presented here demonstrate that the natural substrate, eIF2alpha, also binds to PKR in vitro and interacts with the same or an overlapping domain within PKR. A truncated form of eIF2alpha, representing the N-terminal 123 amino acids and containing the regions of homology to pK3, retains the ability to bind PKR. pK3, eIF2alpha, and the truncated form of eIF2alpha all bind to the C-terminus of PKR containing the catalytic domain, but not to the regulatory N-terminus. Variants of pK3 and eIF2alpha, des-(75-78)-K3L (pK3deltaGYID), and des-(80-83)-eIF2alpha (eIF2alphadeltaGYID), from which the conserved amino acids GYID have been deleted, exhibit a decreased ability to interact with PKR. Similarly, the in vitro binding of pK3, eIF2alpha, and the truncated form of eIF2alpha to PKR can be competed with purified pK3 but not with pK3deltaGYID. In addition, the deletion of GYID from eIF2alpha significantly reduces its ability to be phosphorylated by PKR, demonstrating that PKR recognizes its substrate, at least in part through interaction with sequences remote from the phosphorylation site. In summary, we have shown that the region within PKR that interacts with the pseudosubstrate, pK3, is the same region that interacts with the authentic substrate, eIF2alpha. In addition, we have shown that the N-terminal 123 amino acids of eIF2alpha contains structural elements necessary for recognition by PKR. The results pinpoint the GYID motif, shared between pK3 and eIF2alpha and distant from the phosphorylation site, as being important for the interaction of eIF2alpha with PKR, as well as its phosphorylation.


Subject(s)
Eukaryotic Initiation Factor-2/metabolism , Viral Proteins/metabolism , eIF-2 Kinase/metabolism , Amino Acid Sequence , Binding Sites , Binding, Competitive , Catalysis , Electrophoresis, Polyacrylamide Gel , Eukaryotic Initiation Factor-2/chemistry , HeLa Cells , Humans , Molecular Sequence Data , Peptide Fragments/metabolism , Phosphorylation , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Deletion , Sequence Homology, Amino Acid , Sulfur Radioisotopes/metabolism , Viral Proteins/chemistry , eIF-2 Kinase/chemistry
17.
Virology ; 219(1): 321-3, 1996 May 01.
Article in English | MEDLINE | ID: mdl-8623549

ABSTRACT

One of the cellular defense mechanisms against virus infection is mediated by activating the interferon-induced, double-stranded-RNA-activated protein kinase, PKR. Upon activation, PKR phosphorylates and thereby inactivates the protein synthesis initiation factor, elF-2, leading to cessation of protein synthesis. Viruses have evolved diverse strategies to counteract this cellular antiviral response. A majority of these strategies target PKR to prevent its activation. Recently, we showed that simian virus 40 (SV40) large-T antigen reverses PKR-mediated translational inhibition at a step downstream of PKR activation (Rajan et al., J. Virol. 69, 785--795, 1995). In this paper, we present evidence showing that SV40 can restore efficient translation in cells despite the elevated levels of phosphorylated elF-2 alpha resulting from PKR activation. Thus, SV40 large-T-mediated translational rescue occurs at a step downstream of elF-2 alpha phosphorylation.


Subject(s)
Antigens, Polyomavirus Transforming/metabolism , Eukaryotic Initiation Factor-2/metabolism , Animals , Cell Line , Chlorocebus aethiops , Eukaryotic Initiation Factor-2/antagonists & inhibitors , Gene Expression Regulation, Viral , Phosphorylation , Protein Biosynthesis , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , eIF-2 Kinase
18.
J Biol Chem ; 270(43): 25426-34, 1995 Oct 27.
Article in English | MEDLINE | ID: mdl-7592710

ABSTRACT

PKR is an interferon (IFN)-induced serine/threonine protein kinase that regulates protein synthesis through phosphorylation of eukaryotic translation initiation factor-2 (eIF-2). In addition to its demonstrated role in translational control, recent findings suggest that PKR plays an important role in regulation of gene transcription, as PKR phosphorylates I kappa B alpha upon double-stranded RNA treatment resulting in activation of NF-kappa B DNA binding in vitro (Kumar, A., Haque, J., Lacoste, J., Hiscott, J., and Williams, B.R.G. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6288-6292). To further investigate the role of PKR in transcriptional signaling, we expressed the wild type human PKR and a catalytically inactive dominant negative PKR mutant in the murine pre-B lymphoma 70Z/3 cells. Here, we report that expression of wild type PKR had no effect on kappa-chain transcriptional activation induced by lipopolysaccharide or IFN-gamma. However, expression of the dominant negative PKR mutant inhibited kappa gene transcription independently of NF-kappa B activation. Phosphorylation of eIF-2 alpha was not increased by lipopolysaccharide or IFN-gamma, suggesting that PKR mediates kappa gene transcriptional activation without affecting protein synthesis. Our findings further support a transcriptional role for PKR and demonstrate that there are at least two distinct PKR-mediated signal transduction pathways to the transcriptional machinery depending on cell type and stimuli, NF-kappa B-dependent and NF-kappa B-independent.


Subject(s)
Gene Expression Regulation, Neoplastic , Genes, Immunoglobulin , Immunoglobulin kappa-Chains/genetics , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Animals , Base Sequence , Enzyme Induction , Eukaryotic Initiation Factor-2/metabolism , Gene Expression Regulation, Developmental , Humans , Immunoglobulin M/biosynthesis , Lymphoma, B-Cell , Mice , Molecular Sequence Data , Mutation , NF-kappa B/metabolism , Phosphorylation , Precancerous Conditions , Protein Serine-Threonine Kinases/genetics , RNA, Neoplasm/biosynthesis , Receptors, Antigen, B-Cell/biosynthesis , Recombinant Proteins/metabolism , Sequence Deletion , Transcription, Genetic , Tumor Cells, Cultured , eIF-2 Kinase
19.
Eur J Biochem ; 232(1): 28-36, 1995 Aug 15.
Article in English | MEDLINE | ID: mdl-7556162

ABSTRACT

Double-stranded RNA (dsRNA) induces the vascular cell adhesion molecule VCAM-1 to high levels of expression in human umbilical vein endothelial (HUVE) cells. Although VCAM-1 is also induced by the cytokine interleukin 1 beta (IL-1 beta), activation of the dsRNA-activated protein kinase (PKR) occurs only in response to incubation with dsRNA but not with IL-1 beta. Incubation of HUVE cells with the synthetic dsRNA, poly (I).poly (C), activates PKR with increased autophosphorylation, increased phosphorylation of the translation factor eIF2 alpha, and increased activation of the transcription factor NF-kappa B. Promoter analysis in HUVE cells using a VCAM-1 promoter linked to CAT reporter gene demonstrates that poly (I).poly (C) responsiveness resides in the minimal VCAM-1 promoter that contains two NF-kappa B sites, and deletion of the NF-kappa B sites eliminates basal and poly (I).poly (C)-induced CAT activity, supporting the importance of NF-kappa B in the poly (I).poly (C)-mediated induction of VCAM-1. In vitro studies using purified reagents demonstrate that PKR is capable of phosphorylating I kappa B alpha (the inhibitory subunit of NF-kappa B) in a dsRNA-dependent manner. This suggests that phosphorylation of I kappa B alpha by PKR could be an initial step in the activation of NF-kappa B by dsRNA. NF-kappa B is also activated by IL-1 beta in HUVE cells, but this activation occurs without increased PKR autophosphorylation or eIF2 alpha phosphorylation. Poly (I).poly (C) induces VCAM-1 mRNA levels that are dramatically higher and sustained longer than levels induced by IL-1 beta. Although phosphorylation of eIF2 alpha interferes with protein translation, sufficient VCAM-1 mRNA translation occurs in response to poly (I).poly (C) to yield VCAM-1 protein levels that are similar to levels that are induced by IL-1 beta. This suggests that the higher, sustained VCAM-1 mRNA levels that occur in response to incubation with poly (I).poly (C) compensate for the partial translational block resulting from increased eIF2 alpha phosphorylation. These studies indicate that transcriptional and translational regulatory events that occur in response to activation of PKR by dsRNA are important in the regulation of VCAM-1 gene expression in HUVE cells.


Subject(s)
Endothelium, Vascular/metabolism , Protein Serine-Threonine Kinases/metabolism , RNA, Double-Stranded/metabolism , Vascular Cell Adhesion Molecule-1/biosynthesis , Cell Adhesion , Cells, Cultured , Gene Expression Regulation , Humans , RNA, Double-Stranded/chemistry , eIF-2 Kinase
20.
EMBO J ; 14(15): 3828-34, 1995 Aug 01.
Article in English | MEDLINE | ID: mdl-7641700

ABSTRACT

The interferon induced double-stranded RNA-activated kinase, PKR, has been suggested to act as a tumor suppressor since expression of a dominant negative mutant of PKR causes malignant transformation. However, the mechanism of transformation has not been elucidated. PKR phosphorylates translation initiation factor eIF-2 alpha on Ser51, resulting in inhibition of protein synthesis and cell growth arrest. Consequently, it is possible that cell transformation by dominant negative PKR mutants is caused by inhibition of eIF-2 alpha phosphorylation. Here, we demonstrate that in NIH 3T3 cells transformed by the dominant negative PKR mutant (PKR delta 6), eIF-2 alpha phosphorylation is dramatically reduced. Furthermore, expression of a mutant form of eIF-2 alpha, which cannot be phosphorylated on Ser51 also caused malignant transformation of NIH 3T3 cells. These results are consistent with a critical role of phosphorylation of eIF-2 alpha in control of cell proliferation, and indicate that dominant negative PKR mutants transform cells by inhibition of eIF-2 alpha phosphorylation.


Subject(s)
Cell Transformation, Neoplastic , Eukaryotic Initiation Factor-2/metabolism , Protein Serine-Threonine Kinases/metabolism , 3T3 Cells , Animals , Base Sequence , Eukaryotic Initiation Factor-2/biosynthesis , Eukaryotic Initiation Factor-2/physiology , Humans , Luciferases/biosynthesis , Luciferases/genetics , Mice , Mice, Nude , Molecular Sequence Data , Mutation , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Recombinant Fusion Proteins/biosynthesis , Serine/metabolism , Transfection , eIF-2 Kinase
SELECTION OF CITATIONS
SEARCH DETAIL