Biomedical subjects

B R Williams

Publications and source records attributed to B R Williams.

At least 19 recordsLinked to original sources

PubMed2001

ARED: human AU-rich element-containing mRNA database reveals an unexpectedly diverse functional repertoire of encoded proteins.

The adenylate uridylate-rich elements (AREs) mediate the rapid turnover of mRNAs encoding proteins that regulate cellular growth and body response to exogenous agents such as microbes, inflammatory and environmental stimuli. However, the full repertoire of ARE-containing mRNAs is unknown. Here, we explore the distribution of AREs in human mRNA sequences. Computational derivation of a 13-bp ARE pattern was performed using multiple expectation maximization for motif elicitations (MEME) and consensus analyses. This pattern was statistically validated for the specificity towards the 3'-untranslated region and not coding region. The computationally derived ARE pattern is the basis of a database which contains non-redundant full-length ARE-mRNAs. The ARE-mRNA database (ARED; http://rc.kfshrc.edu.sa/ared) reveals that ARE-mRNAs encode a wide repertoire of functionally diverse proteins that belong to different biological processes and are important in several disease states. Cluster analysis was performed using the ARE sequences to demonstrate potential relationships between the type and number of ARE motifs, and the functional characteristics of the proteins.

Adenosine↗

PubMed2000

Negative regulation of CD8+ T cell function by the IFN-induced and double-stranded RNA-activated kinase PKR.

The IFN-induced and dsRNA-activated kinase (PKR) mediates the antiviral and antiproliferative effects of IFN-alpha and IFN-gamma. Despite these findings, PKR:(-/-) mice have no overt immunological phenotype. Here we tested the role of PKR in cellular immunity by determining the induction and elicitation of contact hypersensitivity in PKR:(-/-) mice, a model of T cell-mediated immunity. When compared with wild type, the magnitude of contact hypersensitivity responses in PKR:(-/-) mice were 2-fold higher and of extended duration. This was also observed when naive recipients of immune CD8(+) T cells from sensitized PKR:(-/-) and CD4(+) T cells from sensitized wild-type PKR:(+/+) or PKR:(-/-) mice were challenged with hapten, indicating a regulatory defect intrinsic to the CD8(+) T cell population. Isolated lymph node T cells from PKR:(-/-) mice were hyperproliferative during Con A-mediated stimulation. These results implicate PKR for the first time in the growth control of mature T lymphocytes and give insight into the negative regulation of CD8(+) T cell-mediated immune responses.

Adoptive Transfer↗

PubMed2000

Identification of connective tissue growth factor as a target of WT1 transcriptional regulation.

The Wilms tumor suppressor WT1 has transcription-activating and -suppressing capabilities. WT1-responsive promoters have been described; however, in large part, it remains unclear which potential downstream genes are physiologically relevant and mediate the function of WT1 in tumorigenesis and development. To identify genes regulated by WT1 in vivo, we used a dominant-negative version of WT1 to modulate WT1 activity in a Wilms tumor cell line. Screening oligonucleotide arrays with RNA from these cells uncovered a number of genes whose expression was altered by abrogation of WT1 function. Several of the genes encode members of the CCN family of growth regulators. The promoter of one of these genes, connective tissue growth factor (CTGF), is suppressed by WT1 both in its endogenous location and in reporter constructs. WT1 regulation of CTGF expression is not mediated by previously identified WT1 recognition elements and may therefore involve a novel mechanism. Our results indicate that CTGF is a bona fide target of WT1 transcriptional suppression and likely plays a role in Wilms tumorigenesis and associated disease syndromes.

Antigens, Neoplasm↗

PubMed2000

Characterization of the gene encoding the 100-kDa form of human 2',5' oligoadenylate synthetase.

The 2'-5' oligoadenylate synthetases (OAS) represent a family of interferon (IFN)-induced proteins implicated in the antiviral action of IFN. When activated by double-stranded (ds) RNA, these proteins polymerize ATP into 2'-5' linked oligomers with the general formula pppA(2'p5'A)n, n greater than or = 1. Three forms of human OAS have been described corresponding to proteins of 40/46, 69/71, and 100 kDa. These isoforms are encoded by three distinct genes clustered on chromosome 12 and exhibit differential constitutive and IFN-inducible expression. Here we describe the structural and functional analysis of the gene encoding the large form of human OAS. This gene has 16 exons with exon/intron boundaries that are conserved among the different isoforms of the human OAS family, reflecting the evolutionary link among them. The promoter region of the p100 gene is composed of multiple features conferring direct inducibility not only by IFNs but also by TNF and all-trans retinoic acid. In contrast, the induction of the p100 promoter by dsRNA is indirect and requires IFN type I production.

2',5'-Oligoadenylate Synthetase↗

PubMed2000

A dynamically tuned double-stranded RNA binding mechanism for the activation of antiviral kinase PKR.

A key step in the activation of interferon-inducible antiviral kinase PKR involves differential binding of viral double-stranded RNA (dsRNA) to its two structurally similar N-terminal dsRNA binding motifs, dsRBM1 and dsRBM2. We show here, using NMR spectroscopy, that dsRBM1 with higher RNA binding activity exhibits significant motional flexibility on a millisecond timescale as compared with dsRBM2 with lower RNA binding activity. We further show that dsRBM2, but not dsRBM1, specifically interacts with the C-terminal kinase domain. These results suggest a dynamically tuned dsRNA binding mechanism for PKR activation, where motionally more flexible dsRBM1 anchors to dsRNA, thereby inducing a cooperative RNA binding for dsRBM2 to expose the kinase domain.

Amino Acid Sequence↗

PubMed2000

Characterization of a 500-kb contig spanning the region between c-Ha-Ras and MUC2 on chromosome 11p15.5.

The 11p15.5 region is associated with a broad range of diseases, including childhood acute myeloid leukemia; non-small cell lung carcinoma; arthrogryposis multiplex congenita, distal type 2B; and bladder cancer. Since targets for these diseases are unknown, we have constructed a physical map consisting of BAC and PAC clones spanning the region from the HRAS1 gene to the cluster of mucin genes on 11p15.5. The contig spans approximately 500 kb and includes 13 genes (9 novel), 9 STSs (5 novel), and 1 SNP and builds upon a published physical map spanning the region from the telomere to the HRAS gene. In addition, we expand the mucin gene cluster located on 11p15.5 to include a novel mucin-like gene (MUCDHL) located less than 250 kb telomeric to MUC6. The identification of potential disease genes within an organizational and evolutionary context provides valuable clues to function and as such will benefit our understanding of this region of the genome.

Chromosomes, Artificial, Bacterial↗

PubMed2000

Identification of critical residues required for suppressor of cytokine signaling-specific regulation of interleukin-4 signaling.

Suppressor of cytokine signaling (SOCS) family proteins were originally identified as cytokine-induced negative regulators of cytokine signaling. We show that SOCS-1 and SOCS-3 inhibit interleukin (IL)-4-dependent signal transducer and activator of transcription 6 (Stat6) activation of and subsequent gene induction. By contrast, SOCS-2 and cytokine-inducible Src homology domain 2 (SH2)-containing protein up-regulate these processes. IL-4 initiates transmembrane signaling through two types of receptor complexes comprising the IL-4Ralpha subunit and the associated Janus kinase 1 (Jak1) as common essential components. We demonstrate that both SOCS-1- and SOCS-3-mediated down-regulation of IL-4 signaling is due to an inhibition of the receptor associated Jak1 activity. The SOCS proteins contain an amino-terminal region of variable length and primary structure, a central SH2 domain, and a carboxyl-terminal conserved motif termed SOCS-box. We show that the SH2 domains of SOCS-2, SOCS-3, and cytokine-inducible SH2-containing protein are functionally redundant in regulating the IL-4-dependent Jak-Stat signaling. The Pre-SH2 domains of SOCS-2 and SOCS-3 confer the specificity of their regulatory function. Importantly, the Pre-SH2 domain of SOCS-3 alone can inhibit IL-4 signaling. The SH2-proximal 25 amino acids of SOCS-3 are sufficient for this inhibition, and the Thr residue at position 24 and the Phe residue at position 25 are individually indispensable for its inhibitory function. Thus, the Thr-Phe motif in the Pre-SH2 domain plays a critical role in SOCS-3-mediated inhibition of the IL-4-dependent Jak-Stat signaling, likely by regulating the mode of SOCS-Jak interaction.

Animals↗

PubMed2000

The protein kinase PKR is required for p38 MAPK activation and the innate immune response to bacterial endotoxin.

Protein kinase RNA-regulated (PKR) is an established component of innate antiviral immunity. Recently, PKR has been shown to be essential for signal transduction in other situations of cellular stress. The relationship between PKR and the stress-activated protein kinases (SAPKs), such as p38 mitogen-activated protein kinase (MAPK), is not clear. Using embryonic fibroblasts from PKR wild-type and null mice, we established a requirement for PKR in the activation of SAPKs by double-stranded RNA, lipopolysaccharide (LPS) and proinflammatory cytokines. This does not reflect a global failure to activate SAPKs in the PKR-null background as these kinases are activated normally by anisomycin and other physicochemical stress. Activation of p38 MAPK was restored in immortalized PKR-null cells by reconstitution with human PKR. We also show that LPS induction of interleukin-6 and interleukin-12 mRNA is defective in PKR-null cells, and that production of these cytokines is impaired in PKR-null mice challenged with LPS. Our findings indicate, for the first time, that PKR is required for p38 MAPK signaling and plays a potentially important role in the innate response against bacterial endotoxin.

Animals↗

PubMed2000

Central role of double-stranded RNA-activated protein kinase in microbial induction of nitric oxide synthase.

NO synthase 2 (NOS2) is induced in airway epithelium by influenza virus infection. NOS2 induction late in the course of viral infection may occur in response to IFN-gamma, but early in infection gene expression may be induced by the viral replicative intermediate dsRNA through the dsRNA-activated protein kinase (PKR). Since PKR activates signaling pathways important in NOS2 gene induction, we determined whether PKR is a component in the signal transduction pathway leading to NOS2 gene expression after viral infection of airway epithelium. We show that NOS2 gene expression in human airway epithelial cells occurs in response to influenza A virus or synthetic dsRNA. Furthermore, dsRNA leads to rapid activation of PKR, followed by activation of signaling components including NF-kappaB and IFN regulatory factor 1. NOS2 expression is markedly diminished and IFN regulatory factor 1 and NF-kappaB activation are substantially impaired in PKR null cells. Strikingly, NOS2 induction in response to LPS is abolished in PKR null cells, confirming a central role for PKR in the general signaling pathway to NOS2.

Animals↗

PubMed2000

Multiple-technique analytical characterization of a mixture containing chemical-weapons simulant from a munition.

An amber yellow organic liquid was found in a munition shell at Dugway Proving Grounds, UT, USA, that was likely used as a simulant of chemical weapons. The primary analytical techniques to characterize the mixture were gas chromatography-infrared detection-mass spectral detection (GC-IR-MS); liquid chromatography-mass spectrometry (LC-MS); nuclear magnetic resonance (NMR) using the nuclei 1H, 13C and 31P; and gas chromatography-atomic emission detection (GC-AED). Six major phosphorus-containing components were identified and confirmed by at least three techniques, and several additional phosphorus-containing components of lower concentration have been identified by GC-IR-MS and LC-MS. Five major non-phosphorus components, including ethyl acetate, diethyl sulfide and dibutylamine, have been identified by multiple techniques. The major phosphorus compound (23.9+/-0.4 wt.%) was O,O,O-triethyl phosphorothioate (I) and the second most abundant (14.4+/-0.2 wt.%) was O,O,S-triethyl phosphorothioate (III). No VX, G-agent, or pesticide was observed in the sample, although III may be a cholinesterase inhibitor which produces delayed toxic response. III also produces a false hit for the pesticide cyanthoate when analyzed by GC-MS-EI. The mixture appears to have been formulated as a chemical warfare agent simulant, most likely as a challenge of agent detection techniques.

Chemical Warfare Agents↗

PubMed2000

Specific phenotypic restoration of an attenuated virus by knockout of a host resistance gene.

To produce disease, viruses must enter the host, multiply locally in host tissues, spread from the site of entry, and overcome or evade host immune responses. At each stage in this infectious process, specific microbial and host genes determine the ultimate virulence of the virus. Genetic approaches have identified many viral genes that play critical roles in virulence and are presumed to target specific components of the host innate and acquired immune response. However, formal proof that a virulence gene targets a specific protein in a host pathway in vivo has not been obtained. Based on cell culture studies, it has been proposed that the herpes simplex virus type 1 gene ICP34.5 (ICP, infected cell protein) enhances neurovirulence by negating antiviral functions of the IFN-inducible double-stranded RNA-dependent protein kinase R or PKR [Chou, J., Chen, J.J., Gross, M. & Roizman, B. (1995) Proc. Natl. Acad. Sci. USA 92, 10516-10520]. Herein, we show that a virus that has been attenuated by deletion of ICP34.5 exhibits wild-type replication and virulence in a host from which the PKR gene has been deleted. We show that restoration of virulence is specific to ICP34.5 and PKR by using additional host and viral mutants. The use of recombinant viruses to infect animals with null mutations in host defense genes provides a formal genetic test for identifying in vivo mechanisms and targets of microbial virulence genes.

Animals↗

PubMed2000

Induction of E-selectin expression by double-stranded RNA and TNF-alpha is attenuated in murine aortic endothelial cells derived from double-stranded RNA-activated kinase (PKR)-null mice.

The adherence of leukocytes on the endothelium is mediated in part by the transient expression of the E-selectin adhesion molecule. Because we have previously shown that the dsRNA-activated kinase PKR mediates dsRNA induction of NF-kappaB, we used murine aortic endothelial (MuAE) cells isolated from wild-type and PKR-null mice to investigate the role of PKR in the induction of E-selectin expression by dsRNA (pIC) and TNF-alpha. E-selectin mRNA and protein expression was inducible by both pIC and TNF-alpha in wild-type MuAE cells, whereas induction of E-selectin expression by these agents was defective in PKR-null MuAE cells. Induction of E-selectin promoter activity and NF-kappaB DNA binding activity were substantially reduced in pIC- or TNF-alpha-treated PKR-null cells, indicating a role for PKR in both pIC and TNF-alpha induction of E-selectin via an NF-kappaB-dependent pathway. In PKR-null cells, pIC-mediated degradation of IkappaBbeta is deficient. Activation of this pathway requires the PKR-dependent degradation of the IkappaBbeta protein. Moreover, both phosphorylated and unphosphorylated activating transcription factor 2 DNA-binding activities were reduced in PKR-null aortic endothelial cells. These results indicate that the PKR is required for full activation of E-selectin expression by pIC and TNF-alpha in primary mouse aortic endothelial cells identifying activating transcription factor 2 as a new target for PKR-dependent regulation and suggest a role for PKR in leukocyte adhesion.

Animals↗

PubMed2000

Regulation of c-myc expression by IFN-gamma through Stat1-dependent and -independent pathways.

Interferons (IFNs) inhibit cell growth in a Stat1-dependent fashion that involves regulation of c-myc expression. IFN-gamma suppresses c-myc in wild-type mouse embryo fibroblasts, but not in Stat1-null cells, where IFNs induce c-myc mRNA rapidly and transiently, thus revealing a novel signaling pathway. Both tyrosine and serine phosphorylation of Stat1 are required for suppression. Induced expression of c-myc is likely to contribute to the proliferation of Stat1-null cells in response to IFNs. IFNs also suppress platelet-derived growth factor (PDGF)-induced c-myc expression in wild-type but not in Stat1-null cells. A gamma-activated sequence element in the promoter is necessary but not sufficient to suppress c-myc expression in wild-type cells. In PKR-null cells, the phosphorylation of Stat1 on Ser727 and transactivation are both defective, and c-myc mRNA is induced, not suppressed, in response to IFN-gamma. A role for Raf-1 in the Stat1-independent pathway is revealed by studies with geldanamycin, an HSP90-specific inhibitor, and by expression of a mutant of p50(cdc37) that is unable to recruit HSP90 to the Raf-1 complex. Both agents abrogated the IFN-gamma-dependent induction of c-myc expression in Stat1-null cells.

Animals↗

PubMed2000

HIV-I TAT inhibits PKR activity by both RNA-dependent and RNA-independent mechanisms.

Replication of the human immunodeficiency virus type 1 (HIV-1) is inhibited by interferons (IFNs), in part through activity of the IFN-inducible protein kinase PKR. To escape this antiviral effect, HIV-1 has developed strategies for blocking PKR function. We have previously shown that the HIV-1 Tat protein can associate with PKR in vitro and in vivo and inhibit PKR activity. Here we present evidence that Tat can inhibit PKR activity by both RNA-dependent and RNA-independent mechanisms. Tat inhibited PKR activation by the non-RNA activator heparin, and also suppressed PKR basal level autophosphorylation in the absence of RNA. However, when Tat and dsRNA were preincubated, the amount of Tat required to inhibit PKR activation by dsRNA depended on the dsRNA concentration. In addition to its function in vitro, Tat can also reverse translation inhibition mediated by PKR in COS cells. The Tat amino acid sequence required for interaction with PKR was mapped to residues 40-58, overlapping the hydrophobic core and basic region of HIV-1 Tat. Alignment of amino acid sequences of Tat and eIF-2alpha indicates similarity between the Tat-PKR binding region and the residues around the eIF-2alpha phosphorylation site, suggesting that Tat and eIF-2alpha may bind to the same site on PKR.

Amino Acid Sequence↗

PubMed2000

Effect of deficiency of the double-stranded RNA-dependent protein kinase, PKR, on antiviral resistance in the presence or absence of ribonuclease L: HSV-1 replication is particularly sensitive to deficiency of the major IFN-mediated enzymes.

Control of viral replication by interferon (IFN) is thought to be principally mediated by the 2',5'-oligoadenylate synthetase (OAS)/RNAse L, double-stranded dependent protein kinase (PKR), and myxovirus resistance protein (Mx) pathways. In this study, we monitored the constitutive and IFN-induced antiviral activity in mouse embryo fibroblasts lines derived from mice with targeted disruption of either PKR or PKR/RNAse L genes. At high multiplicity of infection (moi = 10), the absence of PKR had no effect on replication of vesicular stomatitis virus (VSV) but moderately enhanced encephalomyocarditis virus (EMCV) growth and greatly increased replication of herpes simplex virus-1 (HSV-1). Replication of EMCV, HSV-1, and VSV was modestly higher in PKR-/- RNAse L-/- fibroblasts when compared with control cells. Although the antiviral action of IFN-alpha was unaffected by the absence of PKR, IFN action was significantly impaired in the double knockout cells but was dependent on the stage of the virus cycle. At early stages, it appeared that anti-EMCV and anti-HSV-1 action of IFN-alpha was significantly compromised, although weak residual antiviral activity was seen. The action of IFN-alpha against VSV was specifically compromised at a late stage of virus replication. The results showed that PKR is an important mediator in constitutive resistance against HSV-1 and that RNAse L is also necessary for the full antiviral activity of IFN against a variety of viruses. These results supported the existence of novel pathways aimed toward specific stages of the virus life cycle.

Animals↗

PubMed2000

Kinetic analysis of agonist-induced down-regulation of the beta(2)-adrenergic receptor in BEAS-2B cells reveals high- and low-affinity components.

We examined the interrelationships of internalization and down-regulation of the beta(2)-adrenergic receptor in response to treatment of the BEAS-2B human epithelial cell line with both a series of agonists at high occupancy and with various concentrations of fenoterol that gave occupancies from 0.93 to 0.001. We found that the extent of internalization measured after a 30-min treatment increased as a function of coupling efficiency, with ephedrine, dobutamine, albuterol, fenoterol, and epinephrine giving 0, 7, 17, 48, and 55% internalization, respectively. With the exception of dobutamine, the rates of down-regulation (k(deg)) also showed a dependence on agonist coupling efficiency, giving (in terms of fraction of receptors lost/h) 0.082 with ephedrine, 0.250 with dobutamine, 0.148 with albuterol, 0.194 with fenoterol, and 0.212 with epinephrine. Comparison of down-regulation to internalization showed that weak agonists caused down-regulation in the absence of significant internalization. The extent of internalization caused by fenoterol over a 1000-fold range of occupancy was proportional to agonist occupancy. However, although no internalization was observed with the low concentrations (0.2 and 2 nM fenoterol), these concentrations did cause significant down-regulation. Thus, as with partial agonists, it was clear that down-regulation occurred in the absence of measurable internalization. The kinetics of agonist-induced down-regulation are consistent with a scheme in which down-regulation proceeds by two pathways; a high-affinity, low-capacity component (EC(50) = 0.5 nM) clearly dissociated from internalization and a low-affinity, high-capacity component (EC(50) = 160 nM) closely correlated with internalization.

Adenylyl Cyclases↗

PubMed2000

The B56alpha regulatory subunit of protein phosphatase 2A is a target for regulation by double-stranded RNA-dependent protein kinase PKR.

PKR is a cellular serine/threonine kinase that phosphorylates eukaryotic translation initiation factor 2alpha (eIF2alpha) to regulate protein synthesis. PKR also plays a role in the regulation of transcription, programmed cell death and the cell cycle, processes which likely involve other substrates. In a yeast two-hybrid screen, we isolated human protein phosphatase 2A (PP2A) regulatory subunit B56alpha as a PKR-interacting protein. The interaction between B56alpha and PKR was confirmed by in vitro binding assays as well as by in vivo coimmunoprecipitation, and this interaction is dependent on the catalytic activity of PKR. Moreover, recombinant B56alpha was efficiently phosphorylated by PKR in vitro and an isoelectric point shift in B56alpha was detected in extracts from cells induced with the PKR activator pIC. An in vitro dephosphorylation assay showed that when B56alpha was phosphorylated by PKR, the activity of PP2A trimeric holoenzyme was increased. A functional interaction between B56alpha and PKR was observed in cotransfection assays, where a B56alpha-mediated increase in luciferase expression was inhibited by cotransfection with wild-type PKR. This is likely due to a decreased level of eIF4E phosphorylation caused by an increase in PP2A activity following PKR phosphorylation of B56alpha. Taken together, our data indicate that PKR can modulate PP2A activity by phosphorylating B56alpha to regulate cellular activities.

Animals↗

PubMed2000

Activation of p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase by double-stranded RNA and encephalomyocarditis virus: involvement of RNase L, protein kinase R, and alternative pathways.

Double-stranded RNA (dsRNA) accumulates in virus-infected mammalian cells and signals the activation of host defense pathways of the interferon system. We describe here a novel form of dsRNA-triggered signaling that leads to the stimulation of the p38 mitogen-activated protein kinase (p38 MAPK) and the c-Jun NH(2)-terminal kinase (JNK) and of their respective activators MKK3/6 and SEK1/MKK4. The dsRNA-dependent signaling to p38 MAPK was largely intact in cells lacking both RNase L and the dsRNA-activated protein kinase (PKR), i. e., the two best-characterized mediators of dsRNA-triggered antiviral responses. In contrast, activation of both MKK4 and JNK by dsRNA was greatly reduced in cells lacking RNase L (or lacking both RNase L and PKR) but was restored in these cells when introduction of dsRNA was followed by inhibition of ongoing protein synthesis or transcription. These results are consistent with the notion that the role of RNase L and PKR in the activation of MKK4 and JNK is the elimination, via inhibition of protein synthesis, of a labile negative regulator(s) of the signaling to JNK acting upstream of SEK1/MKK4. In the course of these studies, we identified a long-sought site of RNase L-mediated cleavage in the 28S rRNA, which could cause inhibition of translation, thus allowing the activation of JNK by dsRNA. We propose that p38 MAPK is a general participant in dsRNA-triggered cellular responses, whereas the activation of JNK might be restricted to cells with reduced rates of protein synthesis. Our studies demonstrate the existence of alternative (RNase L- and PKR-independent) dsRNA-triggered signaling pathways that lead to the stimulation of stress-activated MAPKs. Activation of p38 MAPK (but not of JNK) was demonstrated in mouse fibroblasts in response to infection with encephalomyocarditis virus (ECMV), a picornavirus that replicates through a dsRNA intermediate. Fibroblasts infected with EMCV (or treated with dsRNA) produced interleukin-6, an inflammatory and pyrogenic cytokine, in a p38 MAPK-dependent fashion. These findings suggest that stress-activated MAPKs participate in mediating inflammatory and febrile responses to viral infections.

Animals↗