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B R Williams

Publications and source records attributed to B R Williams.

At least 73 records · Page 4Linked to original sources
PubMed1998

Genomic features of human PKR: alternative splicing and a polymorphic CGG repeat in the 5'-untranslated region.

The double-stranded RNA-dependent protein kinase (PKR) is a serine/threonine kinase that plays an important role in the antiviral activities of interferon (IFN). To determine the organization and regulation of the PKR locus, lambda phage and bacterial artificial chromosome (BAC) clones containing the human PKR gene were isolated. Characterization of these clones revealed that PKR has 17 exons and 16 introns dispersed in a genomic region of 50 kb. Sequence analysis of the PKR 5'-flanking region identified a canonical IFN-stimulated response element (ISRE), GAAAACGAAACT. Transient transfection of PKR promoter constructs linked to a luciferase reporter gene into human T98G cells indicated that this 5'-flanking region is capable of functioning as a basal promoter that is also inducible by IFN-alpha and IFN-beta but not IFN-gamma. Interestingly, the PKR gene contains a polymorphic CGG trinucleotide repeat in exon 1. In addition, four PKR alleles, varying in repeat number from 7 to 10, were detected in 30 individual chromosomes. The PKR gene undergoes alternative splicing of exon 2, which gives rise to two forms of 5'-untranslated exons of different length. Although the human and murine PKR proteins have high homology, comparison of their gene structures reveals divergence in 5'-flanking regions, suggesting distinct regulation at the genomic level.

5' Untranslated Regions↗
PubMed1998

Two distinct tumor suppressor loci within chromosome 11p15 implicated in breast cancer progression and metastasis.

Chromosome 11p15 has attracted considerable attention because of the biological importance of this region to human disease. Apart from being an important tumor suppressor locus showing loss of heterozygosity (LOH) in several adult and childhood cancers, 11p15 has been shown by linkage analysis to harbor the gene(s) for the Beckwith-Wiedemann syndrome. Furthermore, the clustering of known imprinted genes in the 11p15.5 region suggests that the target gene may also be imprinted. However, positional cloning efforts to identify the target genes have been complicated by the large size (approximately 10 Mb) and complexity of LOH at 11p15. Here, we have analyzed 94 matched normal and breast tumor samples using 17 polymorphic markers that map to 11p15.5-15.4. We have defined precisely the location of a breast tumor suppressor gene between the markers D11S1318 and D11S4088 (approximately 500 kb) within 11p15.5. LOH at this region occurred in approximately 35-45% of breast tumors analyzed. In addition, we have fine-mapped a second, critical region of LOH, that spans the markers D11S1338-D11S1323 (approximately 336 kb) at 11p15.5-p15.4, that is lost in approximately 55-60% of breast tumors. There is a striking correlation between the loss of the two 11p loci and the clinical and histopathological features of breast tumors. LOH at region 1 correlated significantly (P = 0.016) with early events in malignancy and invasiveness. In contrast, the loss of the more proximal region 2, is highly predictive (P = 0.012) of aggressive metastatic disease. Thus, two distinct tumor suppressor loci on chromosome 11p15 may contribute to tumor progression and metastasis in breast cancer. The fine mapping of this intriguing chromosomal region should facilitate the cloning of the target genes and provide critical clues to understanding the mechanisms that contribute to the evolution of adult and childhood cancers.

Breast↗
PubMed1998

Potential Alu function: regulation of the activity of double-stranded RNA-activated kinase PKR.

Cell stress, viral infection, and translational inhibition increase the abundance of human Alu RNA, suggesting that the level of these transcripts is sensitive to the translational state of the cell. To determine whether Alu RNA functions in translational homeostasis, we investigated its role in the regulation of double-stranded RNA-activated kinase PKR. We found that overexpression of Alu RNA by cotransient transfection increased the expression of a reporter construct, which is consistent with an inhibitory effect on PKR. Alu RNA formed stable, discrete complexes with PKR in vitro, bound PKR in vivo, and antagonized PKR activation both in vitro and in vivo. Alu RNAs produced by either overexpression or exposure of cells to heat shock bound PKR, whereas transiently overexpressed Alu RNA antagonized virus-induced activation of PKR in vivo. Cycloheximide treatment of cells decreased PKR activity, coincident with an increase in Alu RNA. These observations suggest that the increased levels of Alu RNAs caused by cellular exposure to different stresses regulate protein synthesis by antagonizing PKR activation. This provides a functional role for mammalian short interspersed elements, prototypical junk DNA.

Cell Line↗
PubMed1998

How cells respond to interferons.

Interferons play key roles in mediating antiviral and antigrowth responses and in modulating immune response. The main signaling pathways are rapid and direct. They involve tyrosine phosphorylation and activation of signal transducers and activators of transcription factors by Janus tyrosine kinases at the cell membrane, followed by release of signal transducers and activators of transcription and their migration to the nucleus, where they induce the expression of the many gene products that determine the responses. Ancillary pathways are also activated by the interferons, but their effects on cell physiology are less clear. The Janus kinases and signal transducers and activators of transcription, and many of the interferon-induced proteins, play important alternative roles in cells, raising interesting questions as to how the responses to the interferons intersect with more general aspects of cellular physiology and how the specificity of cytokine responses is maintained.

Antiviral Agents↗
PubMed1998

Signal transduction in the interferon system.

Interferons (IFNs) comprise a family of secreted proteins that function as important biologic response modifiers in vertebrates. Interferons are produced in response to viral and other microbial infections, they bind to specific receptors on the surface of target cells, and they induce rapid activation of gene transcription. Interferon-stimulated genes modulate many biologic effects of IFNs. Characterization of the molecular mechanisms of IFN-induced gene activation led to the discovery of two distinct families of proteins, known as the Janus family kinases (Jaks) and the signal transducers and activators of transcription (Stats). These signaling proteins transduce intracellular signals from IFN receptors to the nucleus and activate transcription of IFN-stimulated genes. Subsequent studies have shown that many cytokines, hormones, and growth factors signal their target cells through the Jak-Stat signal transduction pathway. Significant molecular cross-talk between Jaks and Stats recruited to IFN receptors in response to IFN-alpha stimulation and other signaling pathways may be involved in the etiology of some of the side effects associated with IFN-alpha therapy.

Animals↗
PubMed1997

Interferon gamma and interleukin 4 stimulate prolonged expression of inducible nitric oxide synthase in human airway epithelium through synthesis of soluble mediators.

Human respiratory epithelium expresses inducible nitric oxide synthase (iNOS) continuously in vivo, however mechanisms responsible for maintenance of expression are not known. We show that IFNgamma is sufficient for induction of iNOS in primary human airway epithelial cells (HAEC) in vitro, and IL-4 potentiates IFNgamma-induced iNOS expression in HAEC through stabilization of iNOS mRNA. IFNgamma/IL-4- induced iNOS expression in HAEC was delayed in onset and prolonged with expression up to 1 wk. Removal of overlying culture media resulted in loss of expression, while transfer of conditioned media induced iNOS mRNA in other HAEC. IFNgamma and IL-4 stimulation activated STAT1 and STAT6 in HAEC, but conditioned media transfer to HAEC produced even higher levels of STAT1 activation than achieved by direct addition of cytokines. Although cytokine induction of iNOS was dependent on new protein synthesis, conditioned media induction of iNOS in HAEC was not. Further, removal of overlying culture media from cells at different times after cytokine stimulation demonstrated that mediator synthesis and/or secretion important for induction and maintenance of iNOS occurs early after cytokine stimulation. In conclusion, a combination of IFNgamma/ IL-4, which occurs naturally in the lung epithelial lining fluid, leads to maintenance of iNOS expression in human airway epithelium through production of soluble mediators and stabilization of mRNA.

Adolescent↗
PubMed1997

Receptor-associated constitutive protein tyrosine phosphatase activity controls the kinase function of JAK1.

Exposure of cells to protein tyrosine phosphatase (PTP) inhibitors causes an increase in the phosphotyrosine content of many cellular proteins. However, the level at which the primary signaling event is affected is still unclear. We show that Jaks are activated by tyrosine phosphorylation in cells that are briefly exposed to the PTP inhibitor pervanadate (PV), resulting in tyrosine phosphorylation and functional activation of Stat6 (in addition to other Stats). Mutant cell lines that lack Jak1 activity fail to support PV-mediated [or interleukin 4 (IL-4)-dependent] activation of Stat6 but can be rescued by complementation with functional Jak1. The docking sites for both Jak1 and Stat6 reside in the cytoplasmic domain of the IL-4 receptor alpha-chain (IL-4Ralpha). The glioblastoma-derived cell lines T98G, GRE, and M007, which do not express the IL-4Ralpha chain, fail to support Stat6 activation in response to either IL-4 or PV. Complementation of T98G cells with the IL-4Ralpha restores both PV-mediated and IL-4-dependent Stat6 activation. Murine L929 cells, which do not express the gamma common chain of the IL-4 receptor, support PV-mediated but not IL-4-dependent Stat6 activation. Thus, Stat6 activation by PV is an IL-4Ralpha-mediated, Jak1-dependent event that is independent of receptor dimerization. We propose that receptor-associated constitutive PTP activity functions to down-regulate persistent, receptor-linked kinase activity. Inhibition or deletion of PTP activity results in constitutive activation of cytokine signaling pathways.

Amino Acid Sequence↗
PubMed1997

Characterization of the solution complex between the interferon-induced, double-stranded RNA-activated protein kinase and HIV-I trans-activating region RNA.

The antiviral activity of the interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase (PKR) is mediated through dsRNA binding leading to PKR autophosphorylation and subsequent inhibition of protein synthesis. Previous biochemical studies have suggested that autophosphorylation of PKR occurs via a protein-protein interaction and that PKR can form dimers in vitro. Using four independent biophysical and biochemical methods, we have characterized the solution complex formed between PKR and trans-activating region (TAR) RNA, a 57-nucleotide RNA species with double-stranded secondary structure derived from the human immunodeficiency virus type I genome. Chemical cross-linking and gel filtration analyses of PKR.TAR RNA complexes reveals that TAR RNA addition increases PKR dimerization and results in the formation of a solution complex with a molecular weight of approximately 150,000. Addition of TAR RNA to PKR results in a quenching of tryptophan fluorescence, indicative of a conformational shift. Through small angle neutron scattering analysis, we show that PKR exists in solution predominantly as a dimer, and has an elongated solution structure. Addition of TAR RNA to PKR causes a significant conformational shift in the protein at a 2:1 stoichiometric ratio of protein to RNA. Taken together, these data indicate that the PKR activation complex consists of a protein dimer bound cooperatively to one dsRNA molecule.

Base Sequence↗
PubMed1997

A double-stranded RNA-activated protein kinase-dependent pathway mediating stress-induced apoptosis.

Apoptosis occurs in response to different cellular stresses, including viral infection, inflammatory cytokines, growth factor deprivation, and UV light, but it is unclear whether these inducers share a common mechanism of induction. The interferon-induced, double-stranded RNA-activated protein kinase (PKR) has been implicated in processes that rely on apoptosis as control mechanisms in vivo, including antiviral activities, cell growth regulation, and tumorigenesis. Here we report that mouse embryo fibroblasts from mutant mice containing homozygous deletions in the PKR gene (Pkr(0/0) mice) were resistant to apoptotic cell death in response to double-stranded RNA, tumor necrosis factor-alpha, or lipopolysaccharide. The mechanism underlying the suppression of apoptosis in the Pkr(0/0) cells could be attributed to defects in the activation of DNA-binding activity for the transcription factor interferon regulatory factor-1 and in Fas mRNA induction. Thus, these results provide genetic evidence implicating a requirement for PKR in mediating different forms of stress-related apoptosis.

Animals↗
PubMed1997

Deficient cytokine signaling in mouse embryo fibroblasts with a targeted deletion in the PKR gene: role of IRF-1 and NF-kappaB.

The interferon (IFN)-induced double-stranded RNA (dsRNA)-activated Ser/Thr protein kinase (PKR) plays a role in the antiviral and antiproliferative effects of IFN. PKR phosphorylates initiation factor eIF2alpha, thereby inhibiting protein synthesis, and also activates the transcription factor, nuclear factor-kappaB (NF-kappaB), by phosphorylating the inhibitor of NF-kappaB, IkappaB. Mice devoid of functional PKR (Pkr(o/o)) derived by targeted gene disruption exhibit a diminished response to IFN-gamma and poly(rI:rC) (pIC). In embryo fibroblasts derived from Pkr(o/o) mice, interferon regulatory factor 1 (IRF-1) or guanylate binding protein (Gbp) promoter-reporter constructs were unresponsive to IFN-gamma or pIC but response could be restored by co-transfection with PKR. The lack of responsiveness could be attributed to a diminished activation of IRF-1 and/or NF-kappaB in response to IFN-gamma or pIC. Thus, PKR acts as a signal transducer for IFN-stimulated genes dependent on the transcription factors IRF-1 and NF-kappaB.

Animals↗
PubMed1997

Is 'diaphragmatic' attenuation a misnomer?. Evaluation of the anatomic cause of 'diaphragmatic' attenuation in SPECT thallium scanning.

UNLABELLED: The most common artifact on thallium scans in our laboratory has been posterior myocardial attenuation, mostly in males. In the past this has been thought due to the position of the diaphragm. METHODS: To evaluate this concept, matching chest x-rays were examined and measured. Eleven examples of posterior myocardial attenuation were consecutively pulled from our files and compared to eleven consecutive controls. RESULTS: It was found that the diaphragm did not differ significantly. The ratio of the PA diameter of the chest to chest width was significantly (p < 0.03) less than controls. CONCLUSIONS: It is suggested that a male with a flat, wide chest is more prone to posterior myocardial attenuation. If a posterior defect is present in this type of patient it is suggested he be further evaluated to rule out artifact.

Anthropometry↗
PubMed1997

Specific binding of the ETS-domain protein to the interferon-stimulated response element.

Interferon (IFN) activation of genes bearing an IFN-stimulated response element (ISRE) is regulated through binding of IFN-stimulated gene factors (ISGF) to the ISRE found in many IFN-stimulated genes. Using a multimerized human 2-5A synthetase ISRE as probe, we screened lambda gt11 expression libraries for cDNA encoding ISRE-binding activity and isolated a clone for murine proto-oncogene ets-1. The Ets-1 protein binds to the 2-5A synthetase ISRE at a site that also binds ISGF3, a multicomponent factor whose ISRE binding correlates with IFN-induced activation of transcription from ISRE-containing promoters. IFN-induced ISGF3 complex formation on the ISRE can be inhibited by specific Ets-1 antibody. Coexpression of Ets-1 represses ISRE-dependent reporter activity, suggesting that one or more members of the Ets protein family may negatively regulate transcriptional activity mediated by the 2-5A synthetase ISRE.

2',5'-Oligoadenylate Synthetase↗
PubMed1997

Interacting pathways of interferon signaling.

Interferons (IFNs) are important modifiers of biologic response in vertebrate cells. After IFNs are induced, in response to viral or microbial infections, the IFNs bind to specific receptors on the surface of target cells and cause rapid activation of IFN-stimulated genes. These genes mediate the different biologic effects of the IFNs. The molecular characterization of IFN-induced gene activation led to the discovery of the Jak-Stat signal transduction pathway, which is now known to be shared by many other cytokines, growth factors, and hormones. Protein phosphorylation plays a key role in the Jak-Stat pathway, which consists of a cascade of specific protein-protein interactions that culminate in protein-DNA engagement and specific transcription. Although responses in the signaling pathways to various IFN-alpha species have not been examined in detail, it is likely that subtle differences in receptor interaction between the various IFN-alpha species, whether derived from natural or synthetic genes, may produce an unexpected array of biologic effects and clinical responses.

Animals↗
PubMed1996

Regulation of WT1 by phosphorylation: inhibition of DNA binding, alteration of transcriptional activity and cellular translocation.

Phosphorylation is one of the major post-translational mechanisms by which the activity of transcription factors is regulated. We have investigated the role of phosphorylation in the regulation of nucleic acid binding activity and the nuclear translocation of WT1. Two recombinant WT1 proteins containing the DNA binding domain with or without a three amino acid (KTS) insertion (WT1ZF + KTS and WT1ZF - KTS) were strongly phosphorylated by protein kinase A (PKA) and protein kinase C (PKC) in vitro. Both PKA and PKC phosphorylation inhibited the ability of WT1ZF + KTS or WT1ZF - KTS to bind to a sequence derived from the WT1 promoter region in gel mobility shift assays. The binding of WT1ZF - KTS to an EGR1 consensus binding site was also inhibited by prior PKA and PKC phosphorylation. We also demonstrate the RNA binding activity of WT1, but this was not altered by phosphorylation. PKA activation by dibutyryl cAMP in WT1-transfected cells resulted in the reversal of WT1 suppression of a reporter construct. Although WT1 protein is predominantly localized to the nucleus, this expression pattern is altered upon PKA activation, resulting in the cytoplasmic retention of WT1. Accordingly, phosphorylation may play a role in modulating the transcriptional regulatory activity of WT1 through interference with nuclear translocation, as well as by inhibition of WT1 DNA binding.

Amino Acid Sequence↗
PubMed1996

Interferon-alpha-induced phosphorylation and activation of cytosolic phospholipase A2 is required for the formation of interferon-stimulated gene factor three.

Treatment of cells with interferon (IFN)-alpha caused phosphorylation and activation of cytosolic phospholipase A2 (cPLA2). The protein tyrosine kinase Jak1 was found to be necessary for the activation of cPLA2. Jak1 could be co-immunoprecipitated with cPLA2 from cell extracts, indicating that a close physical interaction occurs between these two proteins. The induction of IFN-stimulated gene factor three (ISGF3) by IFN-alpha, is blocked by cPLA2 inhibitors in cell cultures and in cell-free reconstituted systems. However, these inhibitors do not block IFN-alpha or gamma-induced binding of STAT1 to the inverted repeat (IR) element of the IFN regulatory factor 1 (IRF-1) gene. Thus, cPLA2 activations occurs as an early event in the IFN-alpha response and is selectively involved in ISGF3-dependent gene activation.

Base Sequence↗
PubMed1996

Tissue-specific regulation of the WT1 locus.

The 11p13 Wilms' tumor locus consists of two coordinately regulated transcripts, WT1 and WIT-1. These genes are highly expressed in the developing urogenital system, beginning with the urogenital ridge at day 10.5, the metanephric blastema at day 11.5, and during glomerular formation at day 13.5, becoming ultimately restricted to the podocytes. Stromal cells of the gonad also show abundant expression. WT1 is expressed at lower levels in spleen, uterus, mesothelial linings of organs in the abdominal and thoracic cavities, and the ependymal layer of the ventral aspect of the spinal cord. WIT-1 mRNA is about 10-fold less abundant than WT1, but appears to be expressed in the same tissue-restricted manner. Expression of the WT1 protein is required for kidney development, although its physiological function remains to be determined. The function of WIT-1 is similarly unknown but one intriguing possibility is that it is an antisense regulator of WT1. An understanding of events controlling spatial and temporal regulation of these genes will greatly improve our ability to study the role of WT1 and WIT-1 in urogenital development. We have found that while chimeric reporter constructs containing 0.6-2.5 kb of the 5' region of the WT1 gene direct transcription in many different cell lines, we were unable to detect expression in 13.5-day mouse embryos. However, a cosmid containing about 42 kb encompassing this region was able to direct the expression of abundant levels of mRNA from the appropriate transcription initiation sites in both stable transfectants of mouse Leydig cells (TM3) or in transgenic embryos. We are currently localizing the DNA elements required for this expression.

Animals↗
PubMed1996

Analysis of WT1 gene expression during mouse nephrogenesis in organ culture.

The temporal and spatial expression patterns of the Wilms tumor gene, WT1, were studied during the organogenesis of the mouse kidney in vitro. In situ hybridization and immunocytochemistry localized cellular expression of WT1 in whole kidney organ cultures to the induced metanephric mesenchyme and developing podocytes. Organ cultures were further characterized immunocytochemically with antibodies that specifically labeled the different tubular epithelial components and supporting mesenchyme of the developing nephrons. In organ cultures, the WT1 expression pattern could be visualized in induced metanephric mesenchyme and entire cell cohorts of differentiating podocytes. Expression of WT1 and cell specific markers were retained in short-term monolayer cultures of dissociated kidneys. The development of the metanephric kidney in vitro involves a highly restricted temporal and spatial cellular expression pattern of WT1 which closely follows that observed in tissue sections from gestational kidney isolated during organogenesis in the mouse.

Animals↗