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Biomedical subjects

C W Anderson

Publications and source records attributed to C W Anderson.

At least 55 records · Page 3Linked to original sources
PubMed1996

A human RNA polymerase II complex associated with SRB and DNA-repair proteins.

We report here the isolation of a human RNA polymerase II complex containing a subset of the basal transcription factors and the human homologues of the yeast SRB (for suppressors of RNA polymerase B) proteins. The complex contains transcriptional coactivators and increases the activation of transcription. In addition, some components of the RNA polymerase II complex participate in DNA repair.

Chromatography, Affinity↗
PubMed1996

The roles of visual and proprioceptive information during motor program choice in frogs.

Previous studies have shown that leopard frogs, Rana pipiens, use tongue prehension to capture small prey and jaw prehension to capture large prey. After hypoglossal nerve transection, the frogs fail to open their mouths when attempting to feed on small prey, but open their mouths and capture large prey. Here, we investigate how visual information about the prey and proprioceptive information from the tongue interact to influence the motor program choice. Using pieces of earthworm of various sizes, we found that Rana exhibits two different behavior patterns based on prey size. The frogs captured the 1.5-cm prey using tongue prehension, whereas 2.0-cm and larger prey were captured using jaw prehension. After hypoglossal transection, the frogs never opened their mouths when they tried to feed on 1.5-cm prey. When feeding on 3.0-cm and larger prey after transection, they always opened their mouths and captured the prey using jaw prehension. When offered 2.0-cm prey, they alternated randomly between opening and not opening the mouth. Therefore, deafferentation changed the pattern of motor program choice at the behavioral border. This implies that afferents from the tongue interact with visual input to influence motor program choice.

Analysis of Variance↗
PubMed1996

Chemical synthesis of phosphorylated peptides of the carboxy-terminal domain of human p53 by a segment condensation method.

A segment condensation method was developed for the chemical synthesis of large (> 90 amino acid) phosphopeptides and was used to produce phosphorylated and non-phosphorylated derivatives of the C-terminal tetramerization and regulatory domains of human p53 (residues 303-393). Efficient condensation synthesis of the 91 residue p53 domain was achieved in two steps. The non-phosphorylated N-terminal segment p53(303-334) (1) and its derivative phosphorylated at serine 315 (1P315), and the non-phosphorylated middle segment p53(335-360) (2), were synthesized as partially protected peptide thioesters in the solid phase using Boc chemistry. The C-terminal segment p53(361-393) (3) and its derivative phosphorylated at serine 392 (3P392) were synthesized as partially protected peptides in the solid phase using Fmoc chemistry. Phosphoamino acid was incorporated into the N-terminal segment (1P315) at the residue corresponding to p53 serine 315 as Boc-Ser(PO3(Bzl)2)-OH during synthesis. Serine 392 in the C-terminal segment was selectively phosphorylated after synthesis by phosphitylation followed by oxidation. A derivative phosphorylated at serine 378 was synthesized in a one-step condensation of the unphosphorylated N-terminal segment (1) and the phosphorylated long C-terminal segment p53(335-393) (2-3P378). Yields of the ligated peptides after removal of the protecting groups and HPLC purification averaged 60% for the first condensation and 35% for the second condensation. All five p53 peptides exhibited monomer-tetramer association as determined by analytical ultracentrifugation. Circular dichroism spectroscopy revealed that phosphorylation at Ser315 increased the alpha-helical content, which was abolished when Ser392 also was phosphorylated, suggesting an interaction between N-terminal and C-terminal residues of the C-terminal domain of p53.

Amino Acid Sequence↗
PubMed1996

Generality of connotative meaning across methods and subjects.

A test was made of the generality of the connotative meaning scores of evaluation, activity, and potency in a dictionary of the 1000 most frequent words in English that has been used as the basis of a computer system to measure the expression of emotional tone. This was done by correlating those scores with the values for evaluation and activity given for words also found in two independently created dictionaries, one based upon adults' ratings and one upon ratings by children. In replicated findings reported by the authors of the dictionary for adults' responses support for the generality of evaluation scores was more strong than that for activity scores. For the ratings by children the generality of both evaluation and activity dimensions received strong support.

Adult↗
PubMed1996

A quantitative and qualitative assessment of the NOVAWET-Perception bifocal contact lens.

PURPOSE: We prospectively evaluated the NOVAWET-Perception multifocal contact lens for comfort and performance. METHODS: We fit 28 presbyopic patients with this multifocal contact lens. This multifocal rigid gas permeable lens has a multiaspheric back surface and multiple eccentricity values, allowing for a natural field of vision at all angles. Patients were evaluated prior to inclusion in the study and then at 1,3, and 9 months. Evaluations included slit lamp examination, visual acuity, contrast sensitivity, and completion of a questionnaire regarding comfort and overall satisfaction. RESULTS: At the end of the study, 14 of 28 patients (50%) were successfully wearing the NOVAWET-Perception lenses. Of these, 12 (86%) achieved J2 or better near vision and 13 (93%) achieved 20/25 or better distance vision. Fourteen patients failed to complete the study. CONCLUSIONS: We believe the NOVALENS multifocal contact lens is a viable option for the correction of many presbyopic patients.

Contact Lenses↗
PubMed1995

A p53-independent pathway for activation of WAF1/CIP1 expression following oxidative stress.

Incubating human cells in diethylmaleate (DEM) depletes the intracellular pool of reduced glutathione (GSH) and increases the concentration of oxidative free radicals. We found that DEM-induced oxidative stress reduced the ability of p53 to bind its consensus recognition sequence and to activate transcription of a p53-specific reporter gene. Nevertheless, DEM treatment induced expression of WAF1/CIP1 but not GADD45 mRNA. The fact that N-acetylcysteine, a precursor of GSH that blocks oxidative stress, prevented WAF1/CIP1 induction by DEM suggests that WAF1/CIP1 induction probably was a consequence of the ability of DEM to reduce intracellular GSH levels. DEM induced WAF1/CIP1 expression in Saos-2 and T98G cells, both of which lack functional p53 protein. DEM treatment did not produce an increase in membrane-associated protein kinase C, but ERK2, a mitogen-activated protein kinase, was phosphorylated in a manner consistent with ERK2 activation. DEM treatment also produced a dose-dependent delay in cell cycle progression, which at low concentrations (0.25 mM) consisted of a G2/M arrest and at higher concentrations (1 mM) also involved G1 and S phase delays. Our results indicate that oxidative stress induces WAF1/CIP1 expression and arrests cell cycle progression through a mechanism that is independent of p53. This mechanism may provide for cell cycle checkpoint control under conditions that inactivate p53.

Animals↗
PubMed1995

Identification of a gene encoding a yeast histone H4 acetyltransferase.

A collection of yeast temperature-sensitive mutants was screened by an enzymatic assay to find a mutant defective in the acetylation of histone H4. The assay used a fractionated cell extract and measured acetylation of a peptide corresponding to amino acids 1-28 of H4. There are at least two activities in this fraction that acetylate the peptide. A mutation, hat1-1, that eliminates one of the activities was identified and mapped to a locus near the centromere of chromosome XVI. The HAT1 gene was cloned and found to encode a protein of 374 amino acids. Analysis of the peptide used in the assay demonstrated that the HAT1 enzyme acetylates lysine 12 of histone H4. hat1 mutants have no obvious growth defects or phenotypes other than the enzyme defect itself. The HAT1 protein expressed in Escherichia coli gave histone acetyltransferase activity in vitro, demonstrating that HAT1 is the structural gene for the enzyme.

Acetylation↗
PubMed1995

DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product.

DNA-dependent protein kinase (DNA-PK), which is involved in DNA double-stranded break repair and V(D)J recombination, comprises a DNA-targeting component called Ku and an approximately 460 kDa catalytic subunit, DNA-PKcs. Here, we describe the cloning of the DNA-PKcs cDNA and show that DNA-PKcs falls into the phosphatidylinositol (PI) 3-kinase family. Biochemical assays, however, indicate that DNA-PK phosphorylates proteins but has no detectable activity toward lipids. Strikingly, DNA-PKcs is most similar to PI kinase family members involved in cell cycle control, DNA repair, and DNA damage responses. These include the FKBP12-rapamycin-binding proteins Tor1p, Tor2p, and FRAP, S. pombe rad3, and the product of the ataxia telangiectasia gene, mutations in which lead to genomic instability and predisposition to cancer. The relationship of these proteins to DNA-PKcs provides important clues to their mechanisms of action.

Ataxia Telangiectasia↗
PubMed1995

Gene for the catalytic subunit of the human DNA-activated protein kinase maps to the site of the XRCC7 gene on chromosome 8.

The DNA-activated serine/threonine protein kinase (DNA-PK) is composed of a large (approximately 460 kDa) catalytic polypeptide (DNA-PKcs) and Ku, a heterodimeric DNA-binding component (p70/p80) that targets DNA-PKcs to DNA. A 41-kbp segment of the DNA-PKcs gene was isolated, and a 7902-bp segment was sequenced. The sequence contains a polymorphic Pvu II restriction enzyme site, and comparing the sequence with that of the cDNA revealed the positions of nine exons. The DNA-PKcs gene was mapped to band q11 of chromosome 8 by in situ hybridization. This location is coincident with that of XRCC7, the gene that complements the DNA double-strand break repair and V(D)J recombination defects (where V is variable, D is diversity, and J is joining) of hamster V3 and murine severe combined immunodeficient (scid) cells.

Animals↗
PubMed1995

Crystallization and preliminary X-ray diffraction studies of the human adenovirus serotype 2 proteinase with peptide cofactor.

Recombinant human adenovirus serotype 2 proteinase (both native and selenomethionine-substituted) has been crystallized in the presence of the serotype 12, 11-residue peptide cofactor. The crystals (space group P3(1)21 or P3(2)21, one molecule per asymmetric unit, a = b = 41.3 angstrum, c = 197.0 angstrum) grew in solutions containing 20-40% 2-methyl-2,4-pentanediol (MPD), 0.1-0.2 M sodium citrate, and 0.1 M sodium HEPES, pH 5.0-7.5. Diffraction data (84% complete to 2.2 angstrum resolution with Rmerge of 0.0335) have been measured from cryopreserved native enzyme crystals with the Argonne blue (1,024 x 1,024 pixel array) charge-coupled device detector at beamline X8C at the National Synchrotron Light Source (operated by Argonne National Laboratory's Structural Biology Center). Additionally, diffraction data from selenomethionine-substituted proteinase, 65% complete to 2.0 angstrum resolution with Rmerge values ranging 0.05-0.07, have been collected at three X-ray energies at and near the selenium absorption edge. We have determined three of the six selenium sites and are initiating a structure solution by the method of multiwavelength anomalous diffraction phasing.

Adenoviruses, Human↗
PubMed1995

A quantitative and qualitative assessment of the Solitaire Bifocal Contact Lens.

We performed qualitative and quantitative assessments of contact lens performance at 1 and 6 months among 23 patients enrolled in the Solitaire Bifocal Contact Lens Study. Overall satisfaction and comfort at 6 months was rated good to excellent in 83% and 84% of patients, respectively. Daytime acuity at distance and near were rated very good to excellent in 75% and 83% of subjects. Quantitative measurements of distance and near acuities at 1 month revealed 83% and 88% of subjects, respectively, seeing better than or equal to 20/25 or J1. At 6 months there was a moderate decrease in the measured distance acuity. A disparity in glare disability was noted, with 53% of subjects indicating significant glare problems but only 21% of glare tested patients having acuities worse than 20/40. Attrition from the study was highest in the first month and was more common among first-time contact lens users. Overall, this segmented bifocal contact lens is well-tolerated and provides good distance and near vision for presbyopic patients.

Accommodation, Ocular↗
PubMed1994

The DNA-activated protein kinase is required for the phosphorylation of replication protein A during simian virus 40 DNA replication.

The 32-kDa subunit of replication protein A (RPA) is phosphorylated during the S phase of the cell cycle in vivo and during simian virus 40 DNA replication in vitro. To explore the functional significance of this modification, we purified a HeLa cell protein kinase that phosphorylates RPA in the presence of single-stranded DNA. By several criteria we identified the purified enzyme as a form of the DNA-activated protein kinase (DNA-PK), a previously described high molecular weight protein kinase that is capable of phosphorylating a number of nuclear DNA binding proteins. Phosphorylation of RPA by DNA-PK is stimulated by natural single-stranded DNAs but not by homopolymers lacking secondary structure. Studies with the simian virus 40 model system indicate that DNA-PK is required for DNA-replication-dependent RPA phosphorylation. Depletion of the kinase activity, however, has no effect on the extent of DNA replication in vitro. Our data support a model in which phosphorylation of RPA by DNA-PK is activated by formation of replication intermediates containing single- and double-stranded regions. This event may be involved in a signaling mechanism that coordinates DNA replication with the cell cycle.

DNA Repair↗
PubMed1994

DNA-dependent protein kinase is activated by nicks and larger single-stranded gaps.

DNA-PK is a DNA-activated serine/threonine protein kinase capable of phosphorylating a number of nuclear DNA-binding proteins. Purified human DNA-PK has two subunits, a 350-kDa polypeptide, Prkdc, which binds ATP and is presumed to contain the catalytic site, and the Ku autoantigen which mediates DNA binding and activation. Previous studies have shown that DNA-PK is activated in vitro by linear double-stranded DNA fragments; however, the Ku subunit binds a broader range of DNA structures. Here we show that EBP-80, a protein originally isolated as a transcription factor for a retroviral long terminal repeat element and subsequently found to be similar to if not identical with Ku, also mediates kinase activation. The EBP-80-Prkdc complex is activated by nanomolar concentrations of DNA constructs containing single-to-double strand transitions, including a closed stem-loop structure and single strand gaps of 0 (nick), 6, and 30 nucleotides. Kinase activation parallels the ability of EBP-80 to bind these and other constructs. Our results extend the range of DNA configurations known to activate DNA-PK and are consistent with the participation of this enzyme complex in several nuclear functions.

Adenosine Triphosphate↗
PubMed1994

Protein kinases and the response to DNA damage.

Exposure of eukaryotic cells to DNA damaging agents induces gene expression and inhibits cell cycle progression. The first response to damage that has been measured is an increase in protein kinase activity. Several protein kinases are activated in cells exposed to DNA damaging agents, and other kinases that correct damage hypersensitivity mutants have been identified genetically. Current challenges include determining which responses result from damage to DNA and identifying the immediate detectors of damaged DNA that initiate signal transduction.

Animals↗
PubMed1994

The carboxy-terminal serine 392 phosphorylation site of human p53 is not required for wild-type activities.

Wild-type p53 functions in the G1 DNA damage checkpoint pathway by activating gene transcription and preventing cell cycle progression. Others reported that mutation of the serine 386 codon in mouse p53 abolished its ability to suppress growth. Serine 386 of murine p53 and the homologous residue of human p53, serine 392, are phosphorylated in vivo and can be phosphorylated in vitro by casein kinase II (CKII). We constructed mutants that changed serine 392 of human p53 to alanine (p53-S392A) or aspartic acid (p53-S392D); cotransfection of both these mutants with a reporter gene carrying a p53-responsive element into the p53-null Saos-2 cell line activated transcription as well as did wild-type p53. Furthermore, both mutants blocked cell cycle progression after transient transfection in these cells. A stable derivative of the T98G human glioblastoma cell line was established that expressed p53-S392A in response to dexamethasone. Overexpression of this mutant activated transcription of the endogenous waf1 (also called cip1) and mdm2 genes to the same extent as wild-type p53 and also produced growth arrest. Finally, p53-S392A and p53-S392D suppressed foci formation by activated ras and adenovirus E1A oncogenes as efficiently as did wild-type p53. Thus, unlike mutants that altered the serine 15 phosphorylation site, elimination of the serine 392 phosphorylation site had no discernible effect on p53 function. We conclude that neither phosphorylation nor RNA attachment to serine 392 are required for human p53's ability to suppress cell growth or to activate transcription in vivo.

Adenovirus E1A Proteins↗
PubMed1993

The carboxyl-terminal transactivation domain of human serum response factor contains DNA-activated protein kinase phosphorylation sites.

The serum response factor (SRF) is a 67-kDa phosphoprotein that, together with auxiliary factors, modulates transcription of immediate early genes containing serum response elements in their promoters. Here we show that the carboxyl-terminal domain of human SRF is phosphorylated in vivo and is recognized in vitro by the double-stranded DNA-activated serine/threonine-specific protein kinase, DNA-PK. SRF phosphorylation by DNA-PK was stimulated by its cognate binding site. Protein microsequence analysis of a 22-amino acid synthetic SRF peptide and phosphopeptide analysis of genetically altered glutathione S-transferase-SRF fusion proteins identified Ser-435 and Ser-446 of human SRF as sites phosphorylated by DNA-PK. Both serines are followed by glutamine. Changing Gln-436 and Gln-447 to other residues reduced or eliminated phosphorylation by DNA-PK, confirming that these glutamines are important determinants for kinase recognition. The carboxyl-terminal transcription activation domain was mapped within a 71-amino acid region that contains both DNA-PK phosphorylation sites. Amino acid substitutions that interfered with phosphorylation by DNA-PK at Ser-435/446 in GAL4-SRF fusion proteins were reduced in transactivation potency. From these data we suggest that DNA-PK phosphorylation may modulate SRF activity in vivo.

3T3 Cells↗
PubMed1993

Dimerization and DNA binding alter phosphorylation of Fos and Jun.

Fos and Jun form dimeric complexes that bind to activator protein 1 (AP-1) DNA sequences and regulate gene expression. The levels of expression and activities of these proteins are regulated by a variety of extracellular stimuli. They are thought to function in nuclear signal transduction processes in many different cell types. The role of Fos and Jun in gene transcription is complex and may be regulated in several ways including association with different dimerization partners, interactions with other transcriptional factors, effects on DNA topology, and reduction/oxidation of a conserved cysteine residue in the DNA-binding domain. In addition, phosphorylation has been suggested to control the activity of Fos and Jun. Here we show that phosphorylation of Fos and Jun by several protein kinases is affected by dimerization and binding to DNA. Jun homodimers are phosphorylated efficiently by casein kinase II, whereas Fos-Jun heterodimers are not. DNA binding also reduces phosphorylation of Jun by casein kinase II, p34cdc2 (cdc2) kinase, and protein kinase C. Phosphorylation of Fos by cAMP-dependent protein kinase and cdc2 is relatively insensitive to dimerization and DNA binding, whereas phosphorylation of Fos and Jun by DNA-dependent protein kinase is dramatically stimulated by binding to the AP-1 site. These results imply that different protein kinases can distinguish among Fos and Jun proteins in the form of monomers, homodimers, and heterodimers and between DNA-bound and non-DNA-bound proteins. Thus, potentially, these different states of Fos and Jun can be recognized and regulated independently by phosphorylation.

Animals↗