Biomedical subjects

B R Williams

Publications and source records attributed to B R Williams.

At least 91 records · Page 5Linked to original sources

PubMed1996

Testicular cancer in association with developmental renal anomalies and hypospadias.

OBJECTIVES: To characterize the clinical, pathologic, and genetic aspects of patients with a previously undescribed phenotype of testicular germ cell tumors associated with renal hypoplasia or agenesis and urethral hypospadias. METHODS: Review of clinical and pathologic findings and genetic analysis of constitutional and tumor DNA for mutations of the Wilms' tumor suppressor gene (WT1). RESULTS: Clinical findings suggest that this phenotype is distinct from other syndromes associated with renal anomalies and that the associated testicular tumors are histologically and clinically similar to those that occur sporadically. No karyotypic abnormalities, loss of heterozygosity, or mutations in the zinc finger domains (exons 7-10) of WT1 were observed in 5 patients with this phenotype. CONCLUSIONS: The phenotype of testicular germ cell tumor, developmental renal anomalies, and urethral hypospadias constitutes a discrete syndrome caused by a gene distinct from WT1.

Adult↗

PubMed1996

Expression of intracellular interferon constitutively activates ISGF3 and confers resistance to EMC viral infection.

The mechanism(s) by which interferon (IFN)-alpha confers resistance to viruses is currently being characterized. Previous studies have shown that binding of IFN-alpha to its high-affinity receptor activates transcription factor interferon-stimulated gene factor 3 (ISGF3), which positively regulates a number of antiviral genes including 2'-5'-oligoadenylate synthetase (2-5A synthetase). We show that mouse L cells expressing nonsecreted (intracellular) type I human IFN are less susceptible to encephalomyocarditis (EMC) virus infection and have increased levels of 2-5A synthetase. The 2-5A synthetase promoter is constitutively induced, and the antiviral effects are most likely mediated through activation of ISGF3, which occurs constitutively in cell lines expressing intracellular interferon. These data suggest that the internalization of IFN-alpha may play a role in the antiviral properties associated with IFN.

2',5'-Oligoadenylate Synthetase↗

PubMed1996

The realities of empowering teams: a case study.

This case describes the transition of a manufacturing unit from operating under a rigid hierarchical environment to being run by empowered process management teams. This article traces the evolution of the transformation from the early days of skepticism and nonparticipation to the present, where most of the teams function with a large degree of autonomy and have made significant improvements in quality, lead time, and cost. Elements that help the team process are described, and potential hazards are outlined.

Decision Making, Organizational↗

PubMed1996

Overexpression of mouse p140 subunit of replication factor C accelerates cellular proliferation.

Screening of a murine cDNA expression library with an IFN-stimulated response element (ISRE), as a recognition site DNA probe, resulted in the isolation of a cDNA encoding a polypeptide of 1145 amino acids designated ISRE-binding factor-1. This was subsequently shown to be identical to the M(r) 140,000 subunit of replication factor C (RFC). RFC is required, along with the proliferating cell nuclear antigen and DNA polymerase delta, for the synthesis of the leading strand during DNA replication. RFC exhibits a structure-specific DNA-binding activity that has been localized to its M(r) 140,000 subunit (p140). Sequence-specific binding of this polypeptide to the ISRE occurs only with low affinity. Based on DNA-binding activity of the truncated RFC-p140 encoded by the partial cDNA isolated, the DNA-binding domain of this polypeptide was mapped to a region encoded by amino acids 366 to 540. Transfection of NIH 3T3 cells with an expression plasmid containing murine RFC-p140 driven by cytomegalovirus early promoter led to the establishment of stable cell lines that expressed a 2.5- to 3.0-fold higher RFC-p140 protein level in comparison with control cells. The stable clones exhibited significantly accelerated cell proliferation, indicating that RFC-p140 is the limiting subunit of an active RFC complex in normal cells.

3T3 Cells↗

PubMed1995

Human PKR transfected into murine cells stimulates expression of genes under control of the HIV1 or HTLV-I LTR.

We have analyzed the effect of transfection into murine NIH/3T3 cells of the human dsRNA-activated kinase PKR on the expression of the beta-galactosidase reporter gene, placed under control of the HIV1 or the HTLV-I LTR. beta-Galactosidase expression is stimulated when the reporter plasmids are cotransfected with wild-type PKR but inhibited when cotransfected with a catalytically inactive mutant PKR. In the case of HIV1, beta-galactosidase expression was not stimulated when cotransfection was carried out with PKR harboring mutations in the dsRNA binding domains, indicating that stimulation depends on the classical mode of PKR activation through dsRNA binding. In contrast, the dsRNA binding mutants of PKR could still partially stimulate beta-galactosidase expression from the HTLV-I LTR, suggesting that PKR activation in this case may involve different/additional mechanisms. These results show that, in addition to the known down-regulation of protein synthesis through elF2 phosphorylation, PKR can also positively stimulate gene expression in vivo, most probably through phosphorylation of a substrate distinct from elF2.

3T3 Cells↗

PubMed1995

Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase.

Double-stranded RNA-dependent protein kinase (PKR) has been implicated in interferon (IFN) induction, antiviral response and tumor suppression. We have generated mice devoid of functional PKR (Pkr%). Although the mice are physically normal and the induction of type I IFN genes by poly(I).poly(C) (pIC) and virus is unimpaired, the antiviral response induced by IFN-gamma and pIC was diminished. However, in embryo fibroblasts from Pkr knockout mice, the induction of type I IFN as well as the activation of NF-kappa B by pIC, were strongly impaired but restored by priming with IFN. Thus, PKR is not directly essential for responses to pIC, and a pIC-responsive system independent of PKR is induced by IFN. No evidence of the tumor suppressor activity of PKR was demonstrated.

Animals↗

PubMed1995

HIV-1 Tat directly interacts with the interferon-induced, double-stranded RNA-dependent kinase, PKR.

We present evidence that the HIV-1 Tat protein and the RNA-dependent cellular protein kinase, PKR, interact with each other both in vitro and in vivo. Using GST fusion chromatography, we demonstrate that PKR, interacts directly with the HIV-1 Tat protein. The region in Tat sufficient for binding PKR maps within amino acids 20 to 72. In in vitro assays, the two-exon form of Tat (Tat 86) was phosphorylated by PKR, while the one exon form of Tat (Tat 72) inhibited PKR autophosphorylation and substrate phosphorylation. The ability of Tat to interact with PKR was demonstrated in both yeast and mammalian cells. Expression of PKR in yeast results in a growth suppressor phenotype which was reversed by coexpression of a one exon form of Tat. Expression of Tat 72 in HeLa cells resulted in direct interaction with PKR as detected by coimmunprecipitation with a Tat antibody. Tat and PKR also form a coimmunoprecipitable complex in cell-free extracts prepared from productively infected T lymphocytes. The interaction of Tat with PKR provides a potential mechanism by which HIV could suppress the interferon system.

Cell Line↗

PubMed1995

Roles of protein-tyrosine phosphatases in Stat1 alpha-mediated cell signaling.

Different Stat proteins are activated through phosphorylation of unique tyrosine residues in response to different cytokines and growth factors. Interferon-gamma activates Stat1 molecules that form homodimers and bind cognate DNA elements. Here we show that treatment of permeabilized cells with 200-500 microM peroxo-derivatives of vanadium, molybdenum, and tungsten results in the accumulation of constitutively phosphorylated Stat1 alpha molecules. In contrast, treatment of permeabilized cells with orthovanadate, vanadyl sulfate, molybdate, and tungstate at the same range of concentrations does not result in the accumulation of activated Stat1 alpha molecules in the absence of ligand. However, these compounds inhibit the inactivation of interferon-gamma-induced DNA-binding activity of Stat1 alpha. A 4-6-h exposure of the permeabilized cells to orthovanadate, molybdate, and tungstate, but not vanadyl sulfate, results in a ligand-independent activation of Stat1 alpha, which is blocked by the inhibition or depletion of NADPH oxidase activity in the cells, indicating that NADPH oxidase-catalyzed superoxide formation is required for the bioconversion of these metal oxides to the corresponding peroxo-compounds. Interestingly, ligand-independent Stat1 alpha activation by peroxo-derivatives of these transition metals does not require Jak1, Jak2, or Tyk2 kinase activity, suggesting that other kinases can phosphorylate Stat1 alpha on tyrosine 701.

Animals↗

PubMed1995

The interferon-inducible double-stranded RNA-activated protein kinase self-associates in vitro and in vivo.

The interferon-inducible double-stranded (ds) RNA-activated protein kinase (PKR) exhibits antiviral, anticellular, and antitumor activities. The mechanisms of its enzymatic activation by autophosphorylation and of the observed transdominant inhibitory phenotype of enzymatically inactive mutants have invoked PKR dimerization. Here we present direct evidence in support of PKR-PKR interaction. We show that radiolabeled PKR can specifically interact with matrix-bound unlabeled PKR in the absence of dsRNA. The self-association activity resides, in part, in the N-terminal region of 170 residues, which also constitutes the dsRNA-binding domain (DRBD). DRBD can bind to matrix-bound PKR or to matrix-bound DRBD. Dimerization of DRBD was directly demonstrated by chemical crosslinking. Affinity chromatography and electrophoretic mobility supershift assays demonstrated that mutants that fail to bind dsRNA can still exhibit protein-protein interaction. The PKR-PKR interaction could also be observed in a two-hybrid transcriptional activation assay in mammalian cells and consequently is likely to be an important feature of PKR activity in vivo.

Biopolymers↗

PubMed1995

Identification and characterization of human beta 2-chimaerin: association with malignant transformation in astrocytoma.

Molecular processes resulting in the malignant transformation from low- to high-grade astrocytoma remain poorly understood. Using reverse transcriptase PCR, we identified a gene that is differentially expressed in normal brain and low-grade astrocytoma compared to glioblastoma tissues. This gene is identical to human beta 2-chimaerin, which encodes a 468-amino acid GTPase-activating protein for p21rac. The gene was localized to human chromosome 7p15.3 by fluorescence in situ hybridization mapping. Human beta 2-chimaerin is expressed in a variety of human tissues, with the highest expression level detected in human brain and pancreas. RNase protection assays indicated that the expression level of this gene is high in all the normal brain and low-grade astrocytoma samples tested compared to malignant gliomas. The down-regulation of beta 2-chimaerin expression in the high-grade gliomas suggests that decreased expression of this gene may be a feature of progression in the development of malignant glioma.

Amino Acid Sequence↗

PubMed1995

Mutational analysis of the double-stranded RNA (dsRNA) binding domain of the dsRNA-activated protein kinase, PKR.

The interferon-induced, double-stranded RNA (dsRNA)-dependent protein kinase, PKR, is an inhibitor of translation and has antiviral, antiproliferative, and antitumor properties. Previously, the dsRNA binding domain had been located within the N-terminal region of PKR and subsequently shown to include two nearly identical domains comprising residues 55-75 and 145-166. We have undertaken both random and site-directed, alanine-scanning mutagenesis in order to investigate the contribution of individual amino acids within these domains to dsRNA binding. Here we identify 2 residues that were absolutely required for dsRNA binding, glycine 57 and lysine 60. Mutation of 2 other residues within the domain (lysine 64 and leucine 75) resulted in less than 10% binding (compared to wild type). We have also identified a number of other residues that influence dsRNA binding to varying degrees. Mutants that were unable to bind dsRNA were not active in vitro and possessed no antiproliferative activity in vivo. However, dsRNA binding mutants were partially transdominant over wild type PKR in mammalian cells, suggesting that binding of dsRNA activator is not the mechanism responsible for the phenotype of PKR mutants.

3T3 Cells↗

PubMed1995

Analysis of the Wilms tumor suppressor gene WT1 in endometrial carcinoma.

This study reports on the evaluation of the Wilms tumor suppressor gene WT1 in 14 endometrial carcinomas. Despite the fact that several endometrial carcinomas were shown to express WT1, we were unable to demonstrate mutations in the MT1 zinc finger region, suggesting that WT1 does not play a prominent role in the etiology of this malignancy.

Base Sequence↗

PubMed1995

Scaling of hypercapnic ventilatory responsiveness in birds and mammals.

The possible relationship between CO2 responsiveness and body mass in birds was explored using newly acquired ventilatory data from the barn swallow, Hirundo rustica, and the pigeon, Columbia livia, and that from the literature on four other species. Ventilatory responsiveness (% delta V) of birds to 5% inspired CO2 is scaled to body mass to the 0.145 power (% delta V alpha Mb 0.145). A similar allometric relationship exists for data on 7 species of eutherian mammals taken from the literature (% delta V alpha Mb0.130). The The reduced responsiveness to CO2 in small birds and mammals may be related to an elevated hypoxic ventilatory sensitivity, as demonstrated in mammals (Boggs and Tenney, Respir. Physiol. 58: 245-251, 1984). These scaling relationships may reflect a mechanism for minimizing the inhibition of ventilation resulting from excessive loss of CO2 which thereby permits a higher hypoxic ventilatory response in small species. Other mechanisms, however, could include size related differences in mechanics or alveolar ventilation.

Animals↗

PubMed1995

Microsatellite instability at a single locus (D11S988) on chromosome 11p15.5 as a late event in mammary tumorigenesis.

Replication errors at microsatellite repeats are markers for genomic instability in hereditary nonpolyposis colon carcinoma and in some sporadic cancers. Microsatellite sequences may show alterations in one or both alleles in some tumors, suggesting an error in the DNA replication of dinucleotide repeats. We have investigated microsatellite instability (MSI) in sporadic breast tumors at several loci on the short arm of chromosome 11. Among microsatellites studied we found a high frequency of MSI at one specific locus, D11S988 on chromosome 11p15.5. Most colorectal tumors that exhibit MSI display abnormalities of at least one other locus and usually more. By contrast we have detected only one abnormal microsatellite in all the tumors examined. This marker lies between the TH and HBB genes, a subregion previously suggested to harbor a putative tumor suppressor gene for breast cancer. Loss of heterozygosity for chromosome markers at 11p15 has earlier been correlated with poor prognosis. In an unselected panel of primary breast tumors, we observed that 20 of 69 showed mobility shifts of D11S988 in tumor compared with corresponding normal DNA samples. Tumors with instability at D11S988 were rapidly proliferating compared with tumors without MSI. DNA aneuploidy, estrogen receptor positivity and moderate to poorly differentiated tumor phenotype were also characteristics of tumors with MSI at this locus and the majority also exhibited loss of heterozygosity at one or more of the six 11p loci analyzed. Taken together these data suggest that MSI at the D11S988 locus is a late event in mammary tumorigenesis and may be associated with progression of breast carcinomas.

Alleles↗

PubMed1995

In situ expression of the early growth response gene-1 during murine nephrogenesis.

WT1 maps to chromosome 11p13 and encodes a deoxyribonucleic acid (DNA) binding protein whose expression is necessary for normal urogenital development. The WT1 protein binds to some of the same DNA sequences as the early growth response gene-1 (EGR-1) protein, the latter being an immediate-early gene product that activates or represses transcription in a promoter and cell-specific manner. Transient transfection experiments have shown that WT1 can repress EGR-1 activated transcription from the EGR-1 promoter. To determine if WT1 is likely to be a physiologically important repressor of EGR-1 we performed ribonucleic acid (RNA) in situ hybridization of EGR-1 on sequential sagittal sections of murine embryos before and throughout nephrogenesis, and compared the results to our previous study of WT1 expression during murine embryogenesis. Prior to embryological day 9.5 WT1 messenger RNA expression is absent in the embryo proper but is expressed in the maternal uterus. With the initiation of organogenesis on embryological day 10.5 WT1 messenger RNA localizes within the pronephric and mesonephric tissues. By embryological day 11.5 the nephrogenic cord, urogenital ridge and metanephric tissue have WT1 hybridization signals and increasingly centripetal expression of WT1 in the kidney correlates with differentiation from embryological days 11.5 to 16.5. In contrast to previous reports of the tissue restricted expression of WT1, EGR-1 expression by in situ hybridization was apparent in all 3 germ layers and their derivatives throughout embryogenesis. Down-regulation of EGR-1 expression occurred in the maternal uterus as well as the metanephric blastema and its derivatives during renal development. This observation defines a spatial and temporal window during which WT1 competition for EGR-1 DNA binding sites may be involved in regulating EGR-1 expression.

Animals↗

PubMed1995

Expression of the Von Hippel-Lindau tumor suppressor gene, VHL, in human fetal kidney and during mouse embryogenesis.

BACKGROUND: Von Hippel-Lindau (VHL) disease is a familial cancer syndrome that has a dominant inherited pattern which predisposes affected individuals to a variety of tumours. The most frequent tumors are hemangioblastomas of the central nervous system and retina, renal cell carcinoma (RCC), and pheochromocytoma. The recent identification and characterization of the VHL gene on human chromosome 3p and mutational analyses confirms the VHL gene functions as a classical tumor suppressor. Not only are mutations in this gene responsible for the VHL syndrome, but mutations are also very frequent in sporadic RCC. MATERIALS AND METHODS: VHL expression in human kidney and during embryogenesis, was analyzed by in situ mRNA hybridization with 35S-labeled antisense VHL probes, derived from human and mouse cDNAs, on cryosections of human fetal kidney and paraffin sections of murine embryos. RESULTS: In human fetal kidney, there was enhanced expression of VHL within the epithelial lining of the proximal tubules. During embryogenesis, VHL expression was ubiquitous in all three germ cell layers and their derivatives. Expression occurred in the cerebral cortex, midbrain, cerebellum, retina, spinal cord, and postganglionic cell bodies. All organs of the thoracic and abdominal cavities expressed VHL, but enhanced expression was most apparent in the epithelial components of the lung, kidney, and eye. CONCLUSIONS: In human fetal kidney, the enhanced epithelial expression of the VHL gene is consistent with the role of this gene in RCC. There is widespread expression of the VHL gene during embryogenesis, but this is pronounced in areas associated with VHL phenotypes. These findings provide a histological framework for investigating the physiological role of the VHL gene and as basis for further mutational analysis.

Animals↗

PubMed1995

Elevated levels of 2',5'-linked oligoadenylate-dependent ribonuclease L occur as an early event in colorectal tumorigenesis.

RNA decay in IFN-treated cells is controlled by 2'5'-linked oligoadenylate (2-5A)-dependent RNase (RNase L), a uniquely regulated endoribonuclease that requires short 5'-phosphorylated, 2-5A for its activity. Because RNase L is also implicated in the regulation of cell proliferation, we monitored its expression in colorectal adenocarcinomas and noncancerous polyps from familial adenomatous polyposis patients. Elevated levels of RNase L mRNA and activity were found in 17 of 20 tumors compared with corresponding normal mucosa. An mAb against RNase L revealed elevated amounts of this RNase in sections of the tumors, largely in the base of the villi. The occurrence of elevated levels of RNase L seems to be an early event in colorectal tumorigenesis, suggesting that control of RNA turnover is an important step in tumor progression. These data also indicate that regulating RNase L activity may be a useful strategy in treating colorectal carcinomas.

Adenocarcinoma↗

PubMed1994

HIV-1 TAR RNA has an intrinsic ability to activate interferon-inducible enzymes.

The TAR sequence at the 5'-termini of all HIV-1 mRNA species forms a stable structure that is responsible for both transcriptional and translational regulation of HIV-1. Previously we and others reported that purified TAR RNA synthesized by in vitro transcription could activate two interferon-induced enzymes, the protein kinase (PKR) and 2-5A-synthetase. Because the PKR- and 2-5A-systems block protein synthesis initiation and induce RNA decay, respectively, these findings suggested mechanisms for the control of HIV-1 replication by the interferon system. To determine if contaminating dsRNA from in vitro transcription reactions was responsible for this effect, as suggested by Gunnery et al. 1990, (Proc., Natl. Acad. Sci. USA 87, 8687), we have reexamined these findings using chemically synthesized TAR (nucleotides +1 to +57). TAR RNA is shown here to have an intrinsic ability to activate PKR and 2-5A-synthetase. In contrast, a mutant form of TAR designed to have a disrupted secondary structure did not stimulate either enzyme. Chemically synthesized TAR mimicked other dsRNA species in its ability to activate and inhibit PKR at low and high RNA concentrations, respectively. HIV-1 TAT protein inhibited activation of PKR by HIV-1 TAR RNA suggesting an escape mechanism for the virus.

2',5'-Oligoadenylate Synthetase↗