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B L West

Publications and source records attributed to B L West.

At least 37 records · Page 2Linked to original sources
PubMed1992

Synergistic activation of the rat growth hormone promoter by Pit-1 and the thyroid hormone receptor.

The rat GH (rGH) gene is expressed in the pituitary in a highly tissue-specific manner. A pituitary-specific transcription factor, Pit-1 (or GHF-1), and other, more tissue-general factors, including the thyroid hormone receptor (T3R), are important for regulating rGH promoter activity. The relative roles of Pit-1, T3R, and protein kinases in the activation of the rGH promoter were studied. Each component was supplied individually or in combination with the others to human monocyte U937 cells. The transfected rGH promoter was inactive in these cells even when it was cotransfected with either Pit-1 or T3R expression vectors. The rGH promoter carried in a truncated pUC vector could be activated by expression of the T3R if the cells were cultured with inducers of protein kinase-A (forskolin) and protein kinase-C [phorbol 12-myristate 13-acetate (PMA)] activity. By contrast, the PMA- and forskolin-dependent activation of the rGH promoter by Pit-1 expression was comparatively insignificant unless 1) the sequences deleted from the pUC vector (including a putative site for the transcription factor AP1) were restored to the plasmid carrying the rGH promoter; or 2) the T3R was coexpressed, which led to a marked synergistic response. These results indicate the relative inactivity of Pit-1 in isolation from other factors. Activation by forskolin and PMA did not require de novo protein synthesis. The synergistic activation by Pit-1 and the T3R was enhanced, but was not dependent upon, thyroid hormone (T3). The T3-dependent effect operated predominately through a thyroid hormone response element located up-stream of the two Pit-1-binding sites within the rGH promoter, whereas the T3-independent effect did not require any of the known T3R-binding sites on the rGH promoter. These results suggest a role for the more tissue-general T3R and protein kinases in the activation of the rGH promoter. They demonstrate the synergistic interplay between the T3R and Pit-1, underscore the dependence of Pit-1 action on other transcription factors, and implicate Pit-1 as a cofactor, rather than the dominant factor, influencing the tissue-specific expression of the rGH promoter.

Animals↗
PubMed1992

Thyroid hormone alters in vitro DNA binding of monomers and dimers of thyroid hormone receptors.

T3 binds to intranuclear thyroid hormone receptors (TRs) on target DNA elements and exerts profound influences on gene expression by mechanisms not yet characterized. We used gel shift assays and cross-linking experiments to demonstrate that T3 greatly induced the monomeric binding of the hTR beta produced in Escherichia coli to DNA. T3 also increased the gel mobility of these monomer-DNA complexes suggesting they undergo a ligand-induced conformational change. This effect did not depend on the orientation and spacing of the half-site motifs within the DNA structure. In contrast, T3 had diverse effects on the dimeric interaction. T3 increased the dimeric interaction to the palindrome GGTCA.TGACC (an effect lost by spacing the half-sites with 3 base pairs) and decreased the dimeric interaction to the inverted palindrome containing the TGACC.GGTCA motif. Scatchard analyses indicated that the T3 enhancement on binding was due to an increase in the number of TR with high affinity DNA-binding activity and not by increasing the affinity of TR that could bind to DNA. The effects of various T3 analogs were directly related to their affinities for the TR. These ligand effects on in vitro TR-DNA binding may reflect mechanisms by which T3 regulates transcription in vivo.

Animals↗
PubMed1991

Cloning and expression of functional rabbit muscle creatine kinase in Escherichia coli. Addressing the problem of microheterogeneity.

The gene encoding rabbit muscle creatine kinase (CK) has been subcloned into a single plasmid, and the protein expressed in a soluble and functional form in Escherichia coli. The amino terminus, specific activity, and electrophoretic mobility of the E. coli-expressed creatine kinase are all identical with that of creatine kinase purified from rabbit skeletal muscle. Surprisingly, isoelectric focusing shows that the expressed protein displays no less heterogeneity than the tissue-purified material. The identification of the source(s) of this heterogeneity is important for the preparation of highly homogeneous material needed for structural studies and clinical applications. This issue also has implications for studies of the developmental regulation and tissue localization of the various CK genes. Our results allow us to eliminate some of the proposals, such as the presence of multiple alleles, alternative ribosomal initiation sites, and post-translational glycosylation or phosphorylation that have been suggested to explain the presence of the numerous isoforms present in apparently pure preparations of CK.

Animals↗
PubMed1991

Identification of a tumor necrosis factor-responsive element in the tumor necrosis factor alpha gene.

The regulation by tumor necrosis factor alpha (TNF) of its own promoter has been investigated by transient transfection and nuclear protein binding assays. In human K652 erythroleukemia cells TNF produced an 8-10-fold activation of the human TNF promoter linked to the chloramphenicol acetyltransferase gene. The TNF-responsive element was localized to the -125 to -82 region by examining the TNF activation in 5'-deletion or site-directed mutants of the TNF promoter and by demonstrating that the -125 to -82 fragment confers TNF responsiveness to the thymidine kinase promoter. This region contains a palindrome, 5' TGAGCTCA 3', that resembles the consensus binding sequences for the transcription factors, activator protein-1 (AP-1), cyclic AMP-responsive element binding protein (CREB), and activation transcription factor (ATF). An internal deletion in the palindrome abolished the TNF responsiveness, whereas known AP-1 and CREB/ATF elements were unresponsive to TNF. In band shift analyses a nuclear factor from U937 cells specifically bound to the -125 to -82 TNF-responsive fragment in or near the palindromic sequence. Oligonucleotides containing AP-1 or CREB/ATF sites did not effectively compete for the binding, indicating that the U937 cell factor is different from these factors. Anti-c-fos antiserum did not affect binding of the U937 cell factor, whereas anti-c-jun antiserum did block its binding, indicating that either c-jun or a protein antigenically related to c-jun is a component of the factor. These results suggest that the TNF-responsive element is not activated by AP-1 or CREB in U937 cells and that a novel DNA binding factor is important for constitutive and inducible TNF gene expression.

Base Sequence↗
PubMed1991

Productivity: a context for excellence.

Inductively created measures of the sort described above enable the ambulatory care manager to make a crucial decision: Are the outcomes worth the cost involved? As Collins (1988, p. 235) notes, "Our nursing product is quality care based on principles and standards of practice... and substantiated and enhanced through quantifying our practice." By using nursing resources wisely, we can "moderate the march on the market...for caring and access of what we are here for" (Cunningham, 1983, p. 90). In relaying her feelings during treatment for a malignant breast tumor, Kaufman (1989) supports the contention that caring and access are essential parts of a therapeutic regime. Curtin (1987, p. 7) warns that "You cannot provide health care by taking the care out of it." This project allowed nurses to create their own measurement system that identified and quantified the professional nursing tasks involved in each workload indicator. Nonprofessional activities were also identified, quantified, and delegated to appropriate levels of staff, thus increasing the control of the nurses over their own practice environment. In the words of Curtin (1987, p. 7), "Losers count time to make heads roll. Winners use their heads to make time count. This country cannot afford to place its health in the hands of losers."

Ambulatory Care Information Systems↗
PubMed1990

Overlapping Pit-1 and Sp1 binding sites are both essential to full rat growth hormone gene promoter activity despite mutually exclusive Pit-1 and Sp1 binding.

A DNA-binding factor present in a wide variety of cell types is shown to bind to a site (GHF2) within the rat growth hormone gene promoter between nucleotides -147 and -129. Biochemical and transcriptional properties identify this factor as Sp1. The degree of glycosylation of one of the forms of Sp1 is observed to vary in a cell type-specific fashion. The GHF2 binding site overlaps with the more distal (dGHF1) of the two binding sites for the pituitary-specific transcription factor Pit-1. DNase I footprinting and gel retardation analysis with partially purified Sp1 and Pit-1 show that Sp1 and Pit-1 binding to these sites is mutually exclusive. Despite the inability of these two transcription factors to be simultaneously bound to their overlapping sites in vitro, the structural integrity of both binding sites appears essential to the maximal expression of the rat growth hormone promoter after transfection into pituitary GC cells. These findings suggest that activation of the rat growth hormone promoter by Sp1 and Pit-1 may occur through a multistage mechanism.

Animals↗
PubMed1990

Somatotroph- and lactotroph-specific interactions with the homeobox protein binding sites in the rat growth hormone gene promoter.

Nuclear extracts prepared from growth hormone-secreting (GC) and prolactin-secreting (235-1) rat anterior pituitary cell lines were compared for their ability to bind to the DNA sequences conferring tissue-specificity to the expression of the rat growth hormone (rGH) gene promoter. Cell-specific differences in the interaction of Pit-1, a tissue-specific member of the POU-domain transcription factor family, with the pGHF1 binding site were detected by methylation interference experiments; otherwise the Pit-1 proteins present in GC cell and 235-1 cell extracts were similar. Two other protein/DNA complexes, GHF5 and GHF7, were detected by gel mobility shift assays and the binding of both complexes to the rGH promoter depended upon DNA sequences contained within the two binding sites for Pit-1. In contrast to Pit-1 which can bind to either of the two sites independently, a single Pit-1 binding site was insufficient for GHF5 and GHF7 binding; i.e. both Pit-1 binding sites within the rGH promoter were required. Whereas GHF5 was present in nuclear extracts of GC cells and a variety of cells not producing growth hormone, GHF7 binding activity was detected only in the GC cell line (and not in the 235-1 cell line). GHF7 binding activity was therefore more closely correlated with growth hormone gene transcription than was Pit-1. rGH promoters containing mutations which inhibited GHF5, GHF7 and Pit-1 binding were expressed less efficiently than the wild type promoter after transfection into GC cells. One promoter mutation to which the GHF7 complex but not the Pit-1 factor can bind was also transcription deficient demonstrating that Pit-1 binding, independent of GHF7 binding, was nevertheless important to the expression of the rat growth hormone promoter.

Animals↗
PubMed1990

Resolution by diagonal gel mobility shift assays of multisubunit complexes binding to a functionally important element of the rat growth hormone gene promoter.

DNase I footprinting identifies a tissue-general factor, GHF3, binding to the rat growth hormone promoter between nucleotides -239 and -219. Mutation of the GHF3-binding site reduces promoter activity to 30% of that of the wild-type promoter after transfection into GC cells. Southwestern blotting and protein/DNA cross-linking experiments demonstrate that the GHF3-binding factor migrates as a 43-kDa protein. However, multiple GHF3 factor/DNA complexes with different electrophoretic mobilities are detected by gel retardation analysis. A novel technique, the diagonal gel mobility shift assay, is used to demonstrate that five of the different complexes represent multisubunit structures containing a common DNA-binding subunit. In this method, the multisubunit complexes resolved by one-dimensional gel mobility shift assays are observed to partially dissociate during electrophoresis in a second dimension with the DNA-binding subunit detected as a common signal directly below those signals representing the undissociated complexes which lie on a diagonal line. Two of the five complexes also contain an additional subunit in common whereas two other complexes appear to contain completely different subunits interacting with the common DNA-binding subunit. All five complexes copurify during GHF3-binding site-specific DNA affinity chromatography, and this fraction stimulates in vitro transcription in a GHF3-binding site-dependent fashion. Thus, a functionally important region of the rat growth hormone gene promoter interacts with a DNA-binding transcription factor which in turn acts as a docking site for other proteins.

Animals↗
PubMed1990

Removal of a proteolytic activity associated with aggregates formed from expression of creatine kinase in Escherichia coli leads to improved recovery of active enzyme.

Expression of creatine kinase (CK) from a Torpedo californica electric organ cDNA in Escherichia coli results in an insoluble protein product with no detectable CK activity. Although this is a stable aggregate that can be isolated in an enriched form by centrifugation, initial attempts to generate enzyme activity by denaturing and refolding yielded only minute amounts of active protein. We find that these low recoveries are due to proteolysis of the CK during denaturation and refolding. While this proteolytic activity is not inhibited by either phenylmethanesulfonyl fluoride (PMSF) or EDTA, it can be largely removed from the CK aggregate by extraction with a detergent-containing buffer prior to denaturation. This treatment improves the recovery of active CK approximately 100-fold. We have also found similar proteolytic activity associated with the aggregate formed when a mutant of bovine pancreatic trypsin inhibitor (BPTI) is expressed in E. coli. Discovery of this proteolytic activity in two different expression systems suggests that it should be considered as a potential problem for recovery of active protein from other inclusion bodies as well.

Animals↗
PubMed1989

The rat growth hormone gene contains multiple thyroid response elements.

The thyroid hormone receptor exerts transcriptional control over a variety of genes. This report describes four sites that bind this receptor with high affinity within the 5'-flanking DNA of the rat growth hormone gene, approximately centered at -180, -160, -60 and -20 nucleotides from the transcription start site. These sites were defined by gel retardation of short synthetic oligonucleotides using native receptor purified several hundred-fold from rat liver. Binding sites were also defined by methylation interference and methidium-propyl-EDTA footprinting. Alignment of the four binding sites suggests that each contains two purine-rich regions, the more downstream of which, GGGATCGC, is highly conserved. Mutations made within each of the two upstream sites reduce receptor binding affinity. For one mutation, a partial loss of receptor binding strength correlated with a change in electrophoretic mobility, indicating that receptor binding may alter DNA conformation. Mutations at each of the four sites also reduce thyroid hormone responsiveness of the -237/+11 promoter linked to the chloramphenicol acetyltransferase gene coding sequences and transfected into cultured pituitary (GC) cells. These results suggest that several different receptor-binding elements interact to control thyroid hormone responsiveness of the rat growth hormone gene and reveal common sequences that may be important for receptor-DNA recognition.

Animals↗
PubMed1989

Tissue-specific determinants of human atrial natriuretic factor gene expression in cardiac tissue.

Elements controlling tissue-specific expression of the human atrial natriuretic factor gene have been examined in primary cultures of neonatal rat cardiocytes. When a 68-base pair fragment from human atrial natriuretic factor (hANF) 5'-flanking sequence (positions -400 to -333) was placed upstream from the herpes simplex thymidine kinase promoter linked to a bacterial reporter gene (chloramphenicol acetyltransferase), a tissue-specific positive regulatory effect was observed in atrial as well as ventricular cardiocytes but not in nonmyocardial cells. The cis-acting element in this fragment was orientation- and position-dependent. Examination of nuclear protein extracts for the presence of factors capable of interacting with the 5'-flanking sequence of the hANF gene revealed a cardiocyte-specific factor which bound to the 68-base pair fragment. This association was both tissue- and sequence-specific. These findings indicate that a cis-acting element present in the proximal 5'-flanking sequence confers tissue-specific expression upon the hANF gene, possibly through association with a cardiac-specific nuclear protein.

Atrial Natriuretic Factor↗
PubMed1989

Application of behavioral principles in the management of suffocation phobia: a case study of chronic respiratory failure.

This case study presents the first application of behavioral techniques in the management of psychological reactions associated with respiratory weaning. A forty-eight-year-old woman, with a history of brain stem dysfunction and repeated respiratory arrests participated in a four-week behavioral treatment program designed to 1) eliminate suffocation fear and depression, 2) promote respiratory self-sufficiency, and 3) facilitate successful ventilatory weaning. At one year follow-up, the patient continued to display no evidence of respiratory difficulty, depressive symptomatology, or agitated mood states.

Asphyxia↗
PubMed1987

Interaction of a tissue-specific factor with an essential rat growth hormone gene promoter element.

Rat growth hormone (rGH) gene expression is normally restricted to the anterior pituitary. As a model of this tissue specificity, we compared the transient expression of an rGH-chloramphenicol acetyltransferase (CAT) hybrid gene in rGH-producing rat pituitary tumor (GC) cells and in non-rGH-producing rat fibroblast (rat-2) cells. Deletion analysis of the rGH portion of this hybrid gene demonstrated that DNA sequences within 140 base pairs 5' to the rGH gene were sufficient for correct cell type-specific expression. Deletion of an additional 35 base pairs of the rGH 5'-flanking DNA resulted in a loss of expression of the transfected hybrid gene and correlated with the interaction of a putative trans-acting factor with this region of the rGH promoter. This factor was detectable by DNase I footprinting in a crude nuclear extract from GC cells but not from rat-2 cells. Site-directed mutagenesis of the footprint region caused complete loss of expression of a hybrid gene containing 530 base pairs 5' to the rGH gene. Thus, the interaction of this factor, which we term GC2, is likely to be essential for the tissue-specific expression of the rGH gene.

Animals↗
PubMed1987

A pituitary-specific factor interacts with an upstream promotor element in the rat growth hormone gene.

The expression of the rat GH (rGH) gene is limited to the anterior pituitary. Using a DNase I footprinting assay, we have sought to identify specific protein-DNA interactions in the rGH 5'-flanking DNA that may be important in conferring tissue-specific use of the rGH promoter. We have identified a nuclear factor from rGH-secreting GC (rat pituitary) cells which interacts in the rGH 5'-flanking DNA between positions -94 to -62 relative to the start site of rGH gene transcription. This factor, which we have named GC1, is undetectable in nuclear extracts of non-rGH-producing rat fibroblast (Rat-2), or HeLa cells. Mutation of the GC1 binding region in the rGH 5'-flanking DNA results in only a small (30%) decrease in the ability of the rGH promoter to drive the expression of a linked marker gene when transfected into GC cells. GC1 represents, therefore, a novel type of promoter binding factor which is gene-specific and whose distribution is tissue-specific, but which is nonessential for the basal expression of its linked gene. Such a factor may, however, play a role in the modulation of rGH gene expression either through its interaction with other regulatory proteins or ligands or after enzyme modification.

Animals↗
PubMed1984

Creatine kinase protein sequence encoded by a cDNA made from Torpedo californica electric organ mRNA.

Creatine kinase (ATP creatine N-phosphotransferase, EC 2.7.3.2) is important in the maintenance of ATP levels in high energy-requiring tissues such as muscle and brain. A complete understanding of its function requires knowledge of its amino acid sequence. To obtain cDNA clones encoding creatine kinase sequences, a cDNA bank was constructed using mRNA from the electric organ of Torpedo californica and was screened by comparing differential colony hybridization of electric organ and liver-derived 32P-labeled cDNAs. Cloned DNAs have been isolated that can arrest the abundant synthesis of Mr 40,000-43,000 material seen after in vitro translation of electric organ mRNA. One of the clones, CK52g8, was sequenced by the dideoxy M13 method and was found to encode a Mr 42,941 protein, which is 68% homologous to a known partial sequence of rabbit muscle creatine kinase and which has a composition similar to creatine kinases from chicken and rabbit tissues. By contrast, no significant homology was found with the known sequences of kinases that use other substrates. RNA blot hybridization analysis indicated that CK52g8 is complementary to a 1600-base-pair mRNA. Primer extension analysis indicated that CK52g8 is only 5 nucleotides short of a full-length cDNA, implying that it encodes a complete protein sequence. The availability of this complete sequence should be useful in further studies of creatine kinase structure and function using techniques such as site-specific mutagenesis.

Amino Acid Sequence↗
PubMed1983

A method for isolation of intact, translationally active ribonucleic acid.

A method for isolation of large, translationally active RNA species is presented. The procedure involves homogenization of cells or tissues in 5 M guanidine monothiocyanate followed by direct precipitation of RNA from the guanidinium by 4 M LiCl. Modifications are described for use with tissue culture cells, yeast, tissues, or isolated nuclei. The advantages of the procedure include speed, simplicity, avoidance of an ultracentrifugation, and its applicability to large numbers of small samples. The procedure yields large mRNA precursors up to 10 kb and mRNA species which translate very well. However, small (less than 300 nucleotides) RNA species are recovered with a poor yield.

Chemical Precipitation↗