Biomedical subjects

B R Williams

Publications and source records attributed to B R Williams.

At least 145 records · Page 8Linked to original sources

PubMed1991

Regulation of tumor necrosis factor receptor expression by acid-labile interferon-alpha from AIDS sera.

High levels of an unusual acid-labile interferon (IFN) alpha in sera of patients with human immunodeficiency virus (HIV) infection are associated with disease progression to acquired immunodeficiency syndrome (AIDS). Since IFNs have been shown to enhance the cytotoxic actions of tumor necrosis factor (TNF), a potent mediator of inflammation and cachexia, a study was undertaken to investigate whether the acid-labile IFN alpha produced in AIDS can regulate TNF receptor expression. The expression of TNF receptors was determined by studying the interaction of [125I]TNF with cellular receptors. The results show the acid-labile IFN alpha present in AIDS sera is capable of inducing the expression of cellular receptors for TNF. The extent of induction of TNF receptors depends on the concentration of the acid-labile IFN alpha in the AIDS sera. There is no significant induction of TNF receptors when the AIDS sera are preneutralized with polyclonal anti-IFN alpha antibodies. It is also shown that the synthesis of TNF by peripheral blood monocytes (PBM) from patients with HIV infection is enhanced during the progression of HIV infection in vivo. Thus, the TNF system is activated in patients with HIV infection. This activation may be a contributing factor to some of the physiological disturbances including the wasting syndrome observed in AIDS.

Acquired Immunodeficiency Syndrome↗

PubMed1991

Signal transduction and transcriptional regulation of interferon-alpha-stimulated genes.

Interferon-alpha (IFN-alpha) stimulates the expression of a number of genes in a pathway that begins with binding to specific high-affinity plasma membrane receptors. All IFN-alpha-stimulated genes cloned thus far are characterized by the presence of a DNA element, termed Interferon-Stimulated Response Element (ISRE), usually in the 5' upstream region of the genes. The ISRE binds a nuclear factor(s) following IFN-receptor triggered signal transduction and provides a convenient assay for the rapid phase of IFN-alpha signal transduction. This phase utilizes a phospholipase A2-generated second messenger which modulates ISRE-binding factors. Expression cloning has resulted in the identification of two specific ISRE-binding proteins that are candidates as signal recipients. Further advances in our understanding of the molecular mechanisms of IFN action may come through the use of yeast genetics. The human p68 kinase expressed in yeast has a growth inhibitory phenotype and provides a useful alternative system for analyzing components of the IFN-stimulated pathways.

Animals↗

PubMed1991

Functional expression and RNA binding analysis of the interferon-induced, double-stranded RNA-activated, 68,000-Mr protein kinase in a cell-free system.

Eukaryotic viruses have devised numerous strategies to downregulate activity of the interferon-induced, double-stranded (dsRNA)-activated protein kinase (referred to as p68 on the basis of its Mr of 68,000 in human cells). Viruses must exert this control to avoid extensive phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2) by p68 and the resultant negative effects on protein synthesis initiation. To begin to define the molecular mechanisms underlying this regulation, we optimized expression of p68 in an in vitro transcription-translation system utilizing the full-length cDNA clone. The in vitro-expressed kinase was autophosphorylated in response to dsRNAs and heparin in a manner similar to that for the native p68 provided that the kinase inhibitor, 2-aminopurine, was present during the in vitro translation reaction. Further, the activated kinase efficiently phosphorylated its natural substrate, the alpha subunit of eIF-2. Binding experiments revealed that the expressed kinase complexed with the dsRNA activator, reovirus dsRNA, as well as the adenovirus-encoded inhibitor, VAI RNA. Interestingly, both the reovirus RNAs and VAI RNA also complexed with protein kinase molecules that lacked the carboxyl terminus and all catalytic domains. Deletion analysis confirmed that the p68 amino terminus contained critical determinants for reovirus dsRNA and VAI RNA binding. Further, reovirus dsRNA efficiently bound to, but failed to activate, p68 kinase molecules containing a single amino acid substitution in the invariant lysine 295 present in catalytic domain II. Taken together, these data demonstrate that this expression system permits a detailed mutagenic analysis of the regions of p68 required for interaction with virus-encoded activators and repressors.

Adenoviridae↗

PubMed1991

Localization of TIMP in cycling mouse hair.

TIMP (tissue inhibitor of metalloproteinase) is a glycoprotein inhibitor of metalloproteinases that we hypothesize to be involved in the tissue remodeling that occurs during each hair growth cycle. We examined this hypothesis by studying the expression of TIMP at selected times during a single hair cycle using TIMP-lacZ transgenic mice to localize TIMP gene activity in the hair follicle. TIMP gene induction was visualized by staining mouse back skin for beta-galactosidase (beta-gal) activity. Paraffin sections were analyzed for the localization of TIMP expression. TIMP gene activation appears in hair follicles only during the mid-anagen (the growing stage of the hair cycle) primarily in Henle's layer of the inner root sheath. Some expression of TIMP is also seen in a few connective tissue cells, in the sebaceous gland and in cells at the proximity of the dermal papilla cells in catagen (regressing) and telogen (resting) follicles. These results are consistent with a role for TIMP in cyclic remodeling of connective tissue in hair follicles.

Animals↗

PubMed1990

Tissue, developmental, and tumor-specific expression of divergent transcripts in Wilms tumor.

The Wilms tumor locus on chromosome 11p13 has been mapped to a region defined by overlapping, tumor-specific deletions. Complementary DNA clones representing transcripts of 2.5 (WIT-1) and 3.5 kb (WIT-2) mapping to this region were isolated from a kidney complementary DNA library. Expression of WIT-1 and WIT-2 was restricted to kidney and spleen. RNase protection revealed divergent transcription of WIT-1 and WIT-2, originating from a DNA region of less than 600 bp. Both transcripts were present at high concentrations in fetal kidney and at much reduced amounts in 5-year-old and adult kidneys. Eleven of 12 Wilms tumors classified as histopathologically heterogeneous exhibited absent or reduced expression of WIT-2, whereas only 4 of 14 histopathologically homogeneous tumors showed reduced expression. These data demonstrate a molecular basis for the pathogenetic heterogeneity in Wilms tumorigenesis.

Base Sequence↗

PubMed1990

Wilms tumor locus on 11p13 defined by multiple CpG island-associated transcripts.

Wilms tumor is an embryonal kidney tumor involving complex pathology and genetics. The Wilms tumor locus on chromosome 11p13 is defined by the region of overlap of constitutional and tumor-associated deletions. Chromosome walking and yeast artificial chromosome (YAC) cloning were used to clone and map 850 kilobases of DNA. Nine CpG islands, constituting a "CpG island archipelago," were identified, including three islands that were not apparent by conventional pulsed-field mapping, and thus were at least partially methylated. Three distinct transcriptional units were found closely associated with a CpG island within the boundaries of a homozygous DNA deletion in a Wilms tumor.

Chromosome Mapping↗

PubMed1990

Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon.

The double-stranded (ds) RNA-activated protein kinase from human cells is a 68 kd protein (p68 kinase) induced by interferon. On activation by dsRNA in the presence of ATP, the kinase becomes autophosphorylated and can catalyze the phosphorylation of the alpha subunit of eIF2, which leads to an inhibition of the initiation of protein synthesis. Here we report the molecular cloning and characterization of several related cDNAs from which can be deduced the full-length p68 kinase sequence. All of the cDNAs identify a 2.5 kb RNA that is strongly induced by interferon. The deduced amino acid sequence of the p68 kinase predicts a protein of 550 amino acids containing all of the conserved domains specific for members of the protein kinase family, including the catalytic domain characteristic of serine/threonine kinases. In vitro translation of a reconstructed full-length p68 kinase cDNA yields a protein of 68 kd that binds dsRNA, is recognized by a monoclonal antibody raised against the native p68 kinase, and is autophosphorylated.

Amino Acid Sequence↗

PubMed1990

X chromosome inactivation of the human TIMP gene.

X chromosome inactivation results in the cis-limited inactivation of most, but not all, genes on one of the two X chromosomes in mammalian females. The molecular basis for inactivation is unknown. In order to examine the transcriptional activity of human X-linked genes, a series of mouse-human somatic cell hybrids under positive selection for the active or inactive human X chromosome has been created. Northern blot analysis of RNA from these hybrids showed that the human MIC2 gene, which is known to escape X inactivation, was transcribed in hybrids with either the active or inactive X chromosome. In contrast, the human TIMP gene was only transcribed in hybrids with an active human X chromosome. Further analysis using the polymerase chain reaction showed that there was at least one-hundred fold less transcription of the TIMP gene from the inactive X than from the active X chromosome. These findings demonstrate that the human TIMP gene is subject to X inactivation at the level of transcription, and illustrate the usefulness of the polymerase chain reaction to study the extent of X-linked gene repression by the process of X inactivation.

Animals↗

PubMed1990

Molecular characterization of Beckwith-Wiedemann syndrome (BWS) patients with partial duplication of chromosome 11p excludes the gene MYOD1 from the BWS region.

The molecular characterization of two patients with features of Beckwith-Wiedemann syndrome (BWS) and chromosome abnormalities is consistent with the association of this phenotype with a duplication of a portion of chromosome 11. Quantitative Southern blot analysis of DNA from patient A defines a large inherited duplicated segment of chromosome 11. For patient B, a de novo duplication of unknown origin has been shown to contain a segment of 11p15. This chromosome segment includes the genes for insulin-like growth factor 2, beta-hemoglobin, calcitonin A (CALCA), and parathyroid hormone (PTH). However, the myogenic differentiation factor, MYOD1, is not included in the duplicated segment. This demonstrates that MYOD1 is proximal to CALCA and PTH and excludes MYOD1 as the BWS gene. These data place the BWS gene distal to MYOD1 on 11p15.

Beckwith-Wiedemann Syndrome↗

PubMed1990

Definition of the limits of the Wilms tumor locus on human chromosome 11p13.

In a previous report, we described a contiguous restriction map of chromosome band 11p13 that localized the Wilms tumor locus to a small group of NotI fragments. In an effort to identify and isolate the 11p13-associated sporadic Wilms tumor locus, we developed a panel of NotI fragment-specific DNA probes. These probes were selected from genomic libraries constructed using the Chinese hamster ovary-human somatic cell hybrid carrying only human 11p. The libraries were prepared from NotI-digested DNA after size selection by pulsed-field gel electrophoresis. The selected NotI fragments had been previously targeted on the basis of deletion mapping as having a high probability of containing the Wilms tumor locus. We used these newly identified 11p13-specific probes to improve the resolution of the restriction map spanning the Wilms tumor locus. The locus has been defined by a homozygous deletion in a sporadic Wilms tumor. Using these probes, the region of homozygous deletion in this tumor and presumably all or part of the Wilms tumor gene have been confined to two small SfiI fragments spanning less than 350 kb.

Animals↗

PubMed1990

Developmental expression of the endogenous TIMP gene and a TIMP-lacZ fusion gene in transgenic mice.

We determined the expression pattern of the tissue inhibitor of metalloproteinase (TIMP) in the development of the mouse embryo using in situ hybridization and transgenesis. Localized TIMP RNA was first detected at 13.5 days post conceptus (p.c.) in tissues undergoing osteogenesis, such as the mandible, ribs, and calvaria. As development proceeded, TIMP RNA could be detected at additional sites, including the tooth buds, vertebrae, and long bones. To define the sequences regulating TIMP expression, we generated transgenic mice that expressed the Escherichia coli beta-galactosidase gene under control of a 5' region of the mouse TIMP gene containing -2158 to -58 bp upstream of the initiator ATG. By use of an in situ assay for beta-galactosidase activity, the TIMP-lacZ fusion gene product was localized to tissues that also expressed the endogenous TIMP gene, such as the mandible, calvaria, and vertebrae. The localization of TIMP to regions of intramembranous and endochondral bone is similar to that previously reported for TGF-beta, a growth modulator believed to be involved in regulation of extracellular matrix (ECM) formation. Thus, the expression of TIMP in these regions is consistent with it playing a role in ECM deposition and turnover in development.

Animals↗

PubMed1990

The role of interferon and tumor necrosis factor in the pathogenesis of AIDS.

Cytokines including interferon (IFN) and tumor necrosis factor (TNF) are potent modulators of immune processes. They are synthesized in response to microbial infections and inflammation. TNF and IFN interact with other cytokines to elicit differentiation and cellular responses of specific target cells. In view of their multiple biological effects, we have postulated that dysregulation of IFN and TNF may contribute to the pathogenesis of HIV infection. Here, we review data showing that the expression of IFN-alpha receptors is down-regulated in patients with AIDS. As a consequence, HIV-infected cultured cells and cells from AIDS patients show hyporesponsiveness to IFN action. This could contribute to mechanisms by which HIV evades the antiviral activity of IFN-alpha in HIV-infected cells and raise the question of the usefulness of IFN-alpha in the treatment of end-stage AIDS. TNF is a major mediator of inflammation and sepsis and also is capable of inducing the replication of HIV. TNF synthesis and its receptor expression are upregulated by the acid-labile IFN-alpha subtype present in the sera of HIV-infected individuals. In addition, the acid-labile IFN present in AIDS sera may contribute to the pathophysiological changes in sepsis by rendering the cells from AIDS patients hypersensitive to endotoxin stimulation resulting in further synthesis of TNF. Thus aberrant regulation of these cytokines and their cognate receptors are likely contributing factors to the pathogenesis of AIDS.

Acquired Immunodeficiency Syndrome↗

PubMed1989

Regional localization of the TIMP gene on the human X chromosome. Extension of a conserved synteny and linkage group on proximal Xp.

The gene encoding a tissue inhibitor of metallo-proteinases, TIMP, has previously been shown to be X-linked in both the human and mouse genomes. We have used a series of somatic cell hybrids segregating translocation and deletion X chromosomes to map the TIMP gene on the human X chromosome. In combination with previous data, the gene can be assigned to Xp11.23----Xp11.4. Genetic linkage analyses demonstrate that TIMP is linked to the more distal ornithine transcarbamylase (OTC) locus at a distance of about 22 centimorgans. The data are consistent with the conclusion that TIMP maps to a conserved synteny and linkage group on the proximal short arm of the human X chromosome and on the pericentric region of the mouse X chromosome, including loci for synapsin-1, a member of the raf oncogene family, OTC, and TIMP.

Animals↗

PubMed1989

Constitutive expression of a 2',5'-oligoadenylate synthetase cDNA results in increased antiviral activity and growth suppression.

The interferon (IFN)-induced enzyme 2',5'-oligoadenylate (2-5A) synthetase has been implicated in the development of antiviral activity in human and animal cells. However, its role in IFN-mediated growth inhibition remains unclear. To elucidate the function of 2-5A synthetase, we have stably introduced a human 2-5A synthetase cDNA into a human glioblastoma cell line (T98G). Constitutive expression of the cDNA in these cells is associated with increased levels of resistance to infection by encephalomyocarditis virus. One transfected subclone, which expresses elevated levels of 2-5A synthetase enzyme activity, also shows a reduced rate of cellular proliferation.

2',5'-Oligoadenylate Synthetase↗

PubMed1989

Regulation of 2',5'-oligoadenylate synthetase gene expression by interferons and platelet-derived growth factor.

In murine BALB/c 3T3 cell cultures, either beta interferon or platelet-derived growth factor (PDGF) enhanced expression of the 2',5'-oligoadenylate synthetase mRNA and protein. The time course of induction in response to beta interferon was similar to that in response to PDGF. Of several growth factors known to be present in clotted blood serum (i.e., epidermal growth factor, transforming growth factor beta, and PDGF), only PDGF enhanced expression of 2',5'-oligoadenylate synthetase. The linkage of an interferon response element-containing segment from the 5'-flanking region of a human or murine 2',-5'-oligoadenylate synthetase gene made a heterologous gene responsive to interferon. The expression of such a gene construct in transfected cells was also induced by PDGF. Induction by PDGF was inhibited by mono- or polyclonal antibodies to murine interferon, which suggested that induction by PDGF requires interferon. Both PDGF and interferon induced nuclear factors that bound to this interferon response element-containing segment in vitro.

2',5'-Oligoadenylate Synthetase↗

PubMed1988

Interferon-induced binding of nuclear factors to promoter elements of the 2-5A synthetase gene.

Fragments of the 5'-flanking sequence of a human 2-5A synthetase gene were assayed for their ability to respond to interferon-alpha (IFN). Transient transfection assays in monkey cells demonstrated that the 5' boundary of the sequence required for IFN-regulated transcription is, at most, 155 nucleotides upstream from the presumed translational initiation codon. The 3' boundary of this sequence lies within a region of multiple transcription start sites preceded by no obvious TATA box. Binding assays, using a 40-bp probe derived from this IFN-responsive sequence, demonstrated the presence of three IFN-modulated, DNA-factor band shifts using nuclear extracts prepared from human and monkey cells. The induction of these complexes in human cells by IFN occurs with kinetics which closely parallel those previously observed for the transcriptional activation of the 2-5A synthetase gene by IFN. In vivo competition assays showed that the same 40-bp region which bound IFN-modulated factors could decrease the IFN-induced activity of a co-transfected 2-5A synthetase promoter; this fragment, regardless of its orientation, could confer IFN-inducibility on a heterologous promoter.

2',5'-Oligoadenylate Synthetase↗

PubMed1988

Interferon and growth factor modulation of nuclear factors binding to 5' upstream elements of the 2-5A synthetase gene.

We assayed fragments of the 5' flanking sequence of the human 2-5A synthetase gene for their ability to respond to interferon-alpha (IFN) and platelet-derived growth factor (PDGF). Transient transfection assays identified a 40-base pair fragment, which, regardless of orientation, could confer IFN-inducibility on the thymidine kinase promoter. This same fragment was active in monkey and mouse cells and in the latter was responsive to PDGF. The effect of PDGF could be inhibited by anti-interferon antibodies. Gel retardation assays, using the 40-base pair probe, detected the presence of IFN-modulated DNA-binding factors in nuclear extracts from monkey cells. In mouse cells both IFN and PDGF induced the binding of nuclear factors to a synthetic 2-5A synthetase response sequence. Thus, both IFN and growth factors directly or indirectly modulate the binding of nuclear factors to the same region of the 2-5A synthetase gene.

2',5'-Oligoadenylate Synthetase↗

PubMed1988

Interferon and phorbol esters down-regulate sIgM expression by independent pathways.

We studied the effects of recombinant interferon, 12-0-tetradecanoylphorbol-13-acetate (TPA), and phorbol 12, 13 dibutyrate (PDB) on surface immunoglobulin expression by Daudi cells. Incubation of cells with recombinant alpha 2 interferon (IFN-alpha 2) caused a 2.5-fold (60%) decrease in sIgM expression as measured by relative fluorescence index (RFI) using a flow cytometer. This decrease in sIgM expression was independent of inhibitory effects on proliferation and cell cycle progression. TPA or PDB also caused a threefold (67%) decrease in sIgM expression, while enhancing proliferation and cell cycle progression. Coincubation of cells with IFN-alpha 2 and TPA decreased sIgM expression by more than fourfold (greater than 75%), which was greater than the decrease induced by the optimal concentration of either agent alone. Molecular studies demonstrated that the treatment of cells with IFN-alpha 2 or TPA decreased the steady-state levels of mRNA for the heavy chain of IgM (c mu), suggesting that down-regulation of sIgM occurred at a pretranslational level. Activation of the cell membrane sodium/proton antiport did not play an integral role in the IFN-alpha 2 or phorbol-ester-induced pathway of sIgM down-regulation. Whereas IFN-alpha 2 induced an increase in the activity of 2',5'-oligoadenylate (2-5A) synthetase, the addition of TPA to IFN-alpha 2 caused a significant decrease in the activity of this enzyme. Although IFN-alpha 2 and TPA exhibited additive effects on sIgM expression, they had opposing effects on cell proliferation, cell cycle progression, and induction of 2-5A synthetase activity, suggesting that these agents down-regulate sIgM expression through independent pathways.

2',5'-Oligoadenylate Synthetase↗