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Biomedical subjects

C W Anderson

Publications and source records attributed to C W Anderson.

At least 73 records · Page 4Linked to original sources
PubMed1993

Phosphorylation at Ser-15 and Ser-392 in mutant p53 molecules from human tumors is altered compared to wild-type p53.

The product of the p53 gene suppresses cell growth and plays a critical role in suppressing development of human tumors. p53 protein binds DNA, activates transcription, and can be phosphorylated at N- and C-terminal sites. Previously, wild-type p53 was shown to be hyperphosphorylated compared to mutant p53 during p53-mediated growth arrest in vivo. Here we show that Ser-15 and Ser-9 in the N-terminal transactivation domain of wild-type human p53 are phosphorylated in vivo in cells derived from the human glioblastoma line T98G. In [Ile237]p53 and [Ala143]p53, two natural p53 mutants from human tumors that are defective for activation of transcription, phosphorylation at Ser-15 was reduced and phosphorylation at Ser-392 was increased compared to wild-type p53. No change was observed at Ser-9. [His273]p53, a third mutant, had a phosphorylation state similar to that of wild-type p53. We suggest that phosphorylation of Ser-15 may depend on the ability of p53 to adopt a wild-type conformation and may contribute to p53's ability to block cell growth.

Amino Acid Sequence↗
PubMed1993

Viral DNA and a viral peptide can act as cofactors of adenovirus virion proteinase activity.

Human adenovirus (Ad2), like many other viruses, contains a virion-associated proteinase essential for the synthesis of infectious virus particles. We observed proteinase activity in wild-type virus but not in the ts-1 virus, which contains a mutation in the Ad2 L3 endoprotease gene that confers temperature-sensitive processing of virion precursor proteins. Unexpectedly, we did not observe proteinase activity with purified recombinant endoprotease protein (M(r) 23 K). Purified recombinant endoprotease protein, however, complemented the mutation in ts-1 virions, restoring proteinase activity when mixed together. This implied that cofactors may be required. Here we reconstitute proteinase activity in vitro with three purified viral components: (1) the recombinant endoprotease protein; (2) an 11-amino-acid peptide that originates from the carboxy terminus of pVI, the precursor to virion component VI; and (3) adenovirus DNA. The use of DNA for a proteinase activity is unprecedented.

Adenoviruses, Human↗
PubMed1993

Expression and purification of the adenovirus proteinase polypeptide and of a synthetic proteinase substrate.

Simple methods are described for expressing the endoproteinase polypeptides of adenovirus serotypes 2 and 12 in Escherichia coli and for purifying these products from crude bacterial extracts using immobilized metal affinity chromatography. A plasmid for expressing an artificial adenovirus substrate that can be purified by the same method also is described. Cleavage of this substrate by the Ad2 virion proteinase confirmed that the cleavage specificity of the adenovirus proteinase is determined by the four amino acids immediately before the cleavage site. The purified recombinant Ad2 endoproteinase alone was incapable of cleaving the artificial substrate, but cleavage occurred if the artificial substrate was incubated with both recombinant endoproteinase and H2ts1 virions or heat-inactivated wild-type Ad2 virions. These results indicate that, in addition to the 23-kDa proteinase polypeptide, cofactors present in Ad2 virions are required to produce active adenovirus proteinase.

Adenoviruses, Human↗
PubMed1993

Human adenovirus serotype 12 virion precursors pMu and pVI are cleaved at amino-terminal and carboxy-terminal sites that conform to the adenovirus 2 endoproteinase cleavage consensus sequence.

The sequence of a 1158-base pair fragment of the human adenovirus serotype 12 (Ad12) genome was determined. This segment encodes the precursors for virion components Mu and VI. Both Ad12 precursors contain two sequences that conform to a consensus sequence motif for cleavage by the endoproteinase of adenovirus 2 (Ad2). Analysis of the amino terminus of VI and of the peptide fragments found in Ad12 virions demonstrated that these sites are cleaved during Ad12 maturation. This observation suggests that the recognition motif for adenovirus endoproteinases is highly conserved among human serotypes. The adenovirus 2 endoproteinase polypeptide requires additional co-factors for activity (C. W. Anderson, Protein Expression Purif., 1993, 4, 8-15). Synthetic Ad12 or Ad2 pVI carboxy-terminal peptides each permitted efficient cleavage of an artificial endoproteinase substrate by recombinant Ad2 endoproteinase polypeptide.

Adenoviruses, Human↗
PubMed1993

DNA damage and the DNA-activated protein kinase.

DNA-activated protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase that is activated in vitro by DNA fragments. The cellular targets of DNA-PK are nuclear, DNA-binding, regulatory proteins including Sp1, Fos, Jun, Myc, the tumor suppressor protein p53, and RNA polymerase II. These characteristics suggest a role for DNA-PK in coordinating nuclear processes and as a modulator of checkpoint mechanisms activated by DNA damage.

Amino Acid Sequence↗
PubMed1993

A prey-type dependent hypoglossal feedback system in the frog Rana pipiens.

Nerve transection experiments combined with high-speed videography and electromyography were used to characterize a prey-type dependent hypoglossal feedback system which coordinates mouth opening and tongue protraction in the common leopard frog, Rana pipiens. When feeding on small prey, sensory feedback from the tongue through the hypoglossal nerve is necessary to trigger mouth opening. If sensory feedback is prevented from reaching the brain by transection of the hypoglossal nerve, then the mouth fails to open although the feeding behavior appears otherwise normal. However, when feeding on large prey, the mouth opens normally even after the hypoglossal nerve has been transected. Thus, peripheral feedback is not necessary to trigger mouth opening when feeding on large prey, and presumably a central coordination mechanism is used. In Rana pipiens, the evolution of a new tongue morphology and a new motor pattern for feeding on small prey has been accompanied by the evolution of a novel, peripheral mechanism for coordinating tongue protraction and mouth opening. However, the primitive motor pattern for feeding on large prey and the primitive coordinating mechanism have been retained. These results imply that the neural circuits producing the different motor patterns for large and small prey are anatomically distinct at some level in the central nervous system. If they are not anatomically distinct, then sensory feedback should be necessary to trigger mouth opening regardless of which motor pattern is being expressed. While the anatomy of these distinct pathways remains to be elucidated, these results suggest that novel neural circuits may in fact underlie different behaviors even when they seem, superficially, to be relatively similar.

Animals↗
PubMed1993

Mutation of the serine 15 phosphorylation site of human p53 reduces the ability of p53 to inhibit cell cycle progression.

Overexpression of wild-type p53 prevents cells from entering the S phase of the cell cycle. The amino-terminal transactivation region of p53 is phosphorylated by several protein kinases, including DNA-PK, a nuclear serine/threonine protein kinase that in vitro requires DNA for activity. DNA-PK was recently shown to phosphorylate serines 15 and 37 of human p53 (Lees-Miller et al., 1992. Mol. Cell. Biol., 12, 5041-5049). To prevent phosphorylation at these sites, mutants were constructed that changed the codons for serine 15 or serine 37 to alanine codons. Expression of p53-Ala-37 in stably transformed T98G cells blocked progression of the cells into S phase as well as did the expression of wild-type p53. In contrast, p53-Ala-15 was partially defective in blocking cell cycle progression. Several cell clones transformed with the mutant p53-Ala-15 gene expressed normal levels of p53 mRNA but accumulated little or no detectable p53 protein. However, by using a transient expression system driven by a strong cytomegalovirus promoter, we showed that the inability of p53-Ala-15 to fully block cell cycle progression was not due to inadequate levels of expression or to a failure of the mutant protein to accumulate in the nucleus. These results suggest that phosphorylation of Ser-15 may affect p53 function.

Amino Acid Sequence↗
PubMed1992

Human HSP27 is phosphorylated at serines 78 and 82 by heat shock and mitogen-activated kinases that recognize the same amino acid motif as S6 kinase II.

The intracellular concentration of the 27-kDa mammalian heat shock protein, HSP27, increases several-fold after heat and other metabolic stresses and is closely associated with the acquisition of thermotolerance. Posttranslational modifications may also affect the function of HSP27. Heat shock of HeLa cell cultures, or treatment with arsenite, phorbol ester, or tumor necrosis factor, caused a rapid phosphorylation of preexisting HSP27 and the appearance of three phosphorylated isoforms, HSP27 B, C, and D. Digestion with trypsin and fractionation of the peptides by reverse phase high performance liquid chromatography revealed three 32P-labeled phosphopeptides. Microsequence analysis identified peak I as Ala76-Leu77-Ser78-Arg79 and peak II as Gln80-Leu81-Ser82-Ser83-Gly84-Val85- Ser86-Glu87-Ile88-Arg89; peak III contained the undigested peptide pair Ala76-Arg89. Ser82 was the major site and Ser78 the minor site of phosphorylation. Mutant proteins with Ser78 or Ser82 altered to glycine or Ser78-Ser82 double mutants were phosphorylated to reduced extents in vivo after heat or arsenite treatment. Ser78 and Ser82 (and Ser15) occur in the sequence motif RXXS, which is recognized by ribosomal protein S6 kinase II. Mitogenic stimulation of serum-deprived, Go-arrested Chinese hamster cells with serum, thrombin, or fibroblast growth factor also stimulated phosphorylation of HSP27 Ser78 and Ser82, and mitogenic stimulation and heat shock activated protein kinase activities that phosphorylated HSP27 and protein S6 in vitro. These results suggest that HSP27 may exert phosphorylation-activated functions linked with growth signaling pathways in unstressed cells. A homeostatic function at this level could protect cells from adverse effects of signal transduction systems which may be activated inappropriately during stress.

Amino Acid Sequence↗
PubMed1992

Changing trends in the indications for penetrating keratoplasty.

The indications for penetrating keratoplasty have changed over the past several years. Nine hundred ninety-nine penetrating keratoplasty specimens from 1981 through 1990 were analyzed. Overall, keratoconus was the most common indication for penetrating keratoplasty (24.2%). This was followed by pseudophakic bullous keratopathy (23.0%), regraft (13.1%), scarring (8.2%), and Fuchs' endothelial dystrophy (5.8%). Pseudophakic bullous keratopathy was the most common indication for penetrating keratoplasty in each year from 1984 through 1989 with a peak in 1987 (33%). Since 1987, the number of pseudophakic bullous keratopathy cases has decreased; and in 1990, keratoconus (33.1%) surpassed pseudophakic bullous keratopathy (24.1%) as the most common indication for penetrating keratoplasty. This decreasing incidence of pseudophakic bullous keratopathy may reflect the discontinued use of closed-looped anterior chamber and iris-plane intraocular lenses most commonly associated with this complication.

Cataract Extraction↗
PubMed1992

Effect of BW443C81, a novel opioid, on non-cholinergic bronchoconstrictor responses and neurogenic plasma extravasation in the guinea pig.

The novel, peripherally acting opioid peptide, BW443C81, which attenuates airway sensory nerve impulses, was examined on non-cholinergic (NC) constrictor responses in vitro and in vivo and neurogenic plasma extravasation in vivo in guinea-pig airways. Non-cholinergic contractions of guinea pig isolated bronchi, evoked by electrical field stimulation, were concentration-dependently inhibited by BW443C81 and morphine (10 nmol/1-100 mumol/l). In anaesthetised, artificially ventilated guinea pigs, frequency-related NC bronchoconstrictor responses evoked by antidromic electrical stimulation of the vagus nerves were reduced by BW443C81 (100 micrograms/kg/min i.v. infusion) and morphine (1 mg/kg i.v.). Neurogenic plasma extravasation produced by bilateral electrical vagal nerve stimulation in spontaneously breathing, anaesthetised guinea pigs was also inhibited by BW443C81 (1 mg/kg i.v.). The inhibitory effects of BW443C81 were reversed/prevented by naloxone. BW443C81 inhibits NC bronchoconstrictor responses and neurogenic plasma extravasation in guinea pig airways, consistent with its previously described mu-opioid receptor-mediated inhibitory action on airway sensory nerve function.

Anesthesia↗
PubMed1992

The E3-10.4K protein of adenovirus is an integral membrane protein that is partially cleaved between Ala22 and Ala23 and has a Ccyt orientation.

The Ad2 E3-10.4K protein is required together with the E3-14.5K protein to down-regulate the epidermal growth factor receptor in adenovirus-infected cells. Both proteins are also required to prevent tumor necrosis factor cytolysis under certain conditions. 10.4K is a 91 amino acid membrane-associated protein that migrates as two bands, upper and lower, on SDS-PAGE. We show here that the upper band is the primary translation product which initiates at AUG2173 in the E3 transcription unit of Ad2. The upper band is processed slowly (greater than 4 hr to complete) into the lower band by proteolytic cleavage between residues Ala22 and Ala23 by a microsome-associated protease. The upper and lower bands become equal in abundance, after which they are very stable. The N-terminus of the in vivo-derived upper band is not blocked to sequencing and it retains its initiating Met. 10.4K has a hydrophobic domain (H1) near its N-terminus that is probably a signal sequence for membrane insertion; cleavage of this signal is atypical because it was not cotranslational in vivo and it was not complete. 10.4K has a second hydrophobic domain (H2) located within residues 35-60. H2 appears to be a transmembrane (stop transfer) domain because both the upper and the lower 10.4K bands remained associated with membranes after extraction at pH 11.5, because both bands were extracted into the detergent phase with Triton X-114, and because both bands were only partially reduced in size when 10.4K-containing microsomes were digested with proteinase K. These proteinase K-digested bands were immunoprecipitated with an antipeptide antiserum against residues 19-34 but not with an antiserum against residues 68-80 or 77-91, indicating that both 10.4K bands are orientated in the membrane with the C-terminus in the cytoplasm. We conclude that the lower band of 10.4K is a type I bitopic membrane protein and suggest that the upper band is a polytopic membrane protein with both the H1 and the H2 hydrophobic domains spanning the membrane.

Adenovirus Early Proteins↗
PubMed1992

Postoperative inflammation following cataract extraction caused by bacterial contamination of the cleaning bath detergent.

Five of 16 patients who had uncomplicated cataract extraction with intraocular lens (IOL) implantation within a 2.5 day period experienced increased anterior segment inflammation on their first postoperative day. Four of these five patients had phacoemulsification and one a planned extracapsular cataract extraction. All had posterior chamber IOL implantation in the capsular bag. In all patients, this anterior segment inflammation cleared with topical steroids over two to three weeks with no evident residual ocular effects. A careful search for the possible cause of the inflammation showed that the ultrasonic cleaning bath and Weck liquid detergent used to clean the instruments contained Klebsiella pneumoniae bacteria. Further investigation demonstrated the presence of a heat-stable endotoxin produced by the bacteria. We postulate that endotoxin remaining on the instruments after cleaning and sterilization caused this postoperative anterior segment inflammation. To the best of our knowledge, these are the first reported cases caused by contaminated liquid detergent.

Aged↗
PubMed1992

DNA binding provides a signal for phosphorylation of the RNA polymerase II heptapeptide repeats.

Isolated transcription complexes contain a protein kinase that phosphorylates the heptapeptide repeats of the carboxy-terminal domain (CTD) of the RNA polymerase II (RNAP II) large subunit in an apparently promoter-dependent manner. We now show that the essential features of this reaction can be reproduced in a reconstituted system containing three macromolecular components: a fusion protein consisting of the CTD of RNAP II fused to a heterologous DNA-binding domain, an activating DNA fragment containing the recognition sequence for the fusion protein, and a protein kinase that binds nonspecifically to DNA. This kinase closely resembles a previously known DNA-dependent protein kinase. Evidently, the association of the CTD with DNA provides a key signal for phosphorylation. There appears to be no absolute requirement for specific contacts with other DNA-bound transcription factors.

Animals↗
PubMed1992

The adenovirus E3 14.5-kilodalton protein, which is required for down-regulation of the epidermal growth factor receptor and prevention of tumor necrosis factor cytolysis, is an integral membrane protein oriented with its C terminus in the cytoplasm.

We previously reported that the adenovirus type 5 E3 14.5-kilodalton protein (14.5K) forms a complex with E3 10.4K and that both proteins are required to down-regulate the epidermal growth factor receptor in adenovirus-infected human cells. Both proteins are also required to prevent cytolysis by tumor necrosis factor of most mouse cell lines infected by adenovirus mutants that lack E3 14.7K. The E3 14.5K amino acid sequence suggests that 14.5K is an integral membrane protein with an N-terminal signal sequence for membrane insertion. Here we show that 14.5K was found exclusively in cytoplasmic membrane fractions. Radiochemical sequencing of 14.5K indicated that the N-terminal signal sequence is cleaved predominantly between Cys-18 and Ser-19. With a mutant that does not express 10.4K, cleavage occurs predominantly between Phe-17 and Cys-18, indicating that the presence or absence of 10.4K affects the signal cleavage site. 14.5K was extracted into the detergent phase with Triton X-114, it remained associated with membranes after extraction with Na2CO3 at pH 11.5, and it was partially protected by membranes from proteinase K digestion; these observations indicate that 14.5K is an integral membrane protein. Proteinase K digestion followed by immunoprecipitation with antipeptide antisera directed against the N or C terminus of mature 14.5K indicated that 14.5K is oriented in the membrane with its N terminus in the lumen and its C terminus in the cytoplasm. Thus, 14.5K is a type I bitopic membrane protein. Previous studies indicated that 10.4K is also an integral membrane protein oriented with its C terminus in the cytoplasm. Altogether, these findings suggest that cytoplasmic membranes are the site of action when 10.4K and 14.5K down-regulate the epidermal growth factor receptor and prevent tumor necrosis factor cytolysis.

Adenovirus Early Proteins↗
PubMed1992

Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53.

Human DNA-PK is a nuclear, serine/threonine protein kinase that, when activated by DNA, phosphorylates several DNA-binding substrates, including the tumor suppressor protein p53. To identify which p53 residues are phosphorylated, we examined DNA-PK's ability to phosphorylate synthetic peptides corresponding to human p53 sequences. Serines 15 and 37 in the amino-terminal transactivation domain of human p53, and serines 7 and 18 of mouse p53, were phosphorylated by DNA-PK in the context of synthetic peptides. Other serines in these p53 peptides, and serines in other p53 peptides, including peptides containing the serine 315 p34cdc2 site and the serine 392 casein kinase II site, were not recognized by DNA-PK or were phosphorylated less efficiently. Phosphorylation of the conserved serine 15 in human p53 peptides depended on the presence of an adjacent glutamine, and phosphorylation was inhibited by the presence of a nearby lysine. Phosphorylation of recombinant wild-type mouse p53 was inhibited at high DNA concentrations, suggesting that DNA-PK may phosphorylate p53 only when both are bound to DNA at nearby sites. Our study suggests that DNA-PK may have a role in regulating cell growth and indicates how phosphorylation of serine 15 in DNA-bound p53 could alter p53 function.

Amino Acid Sequence↗
PubMed1992

The evolution of neural circuits controlling feeding behavior in frogs.

Our approach to understanding motor systems is a phylogenetic, 'outside-in' approach, the goal of which is to identify behavioral transitions during phylogenesis and elucidate their neurological basis. In this paper, we review the results of recent behavioral, biomechanical and neurological studies on frog feeding behavior. These studies show that highly protrusible tongues have evolved numerous times independently among frogs, and that the biomechanics and neuromuscular control of feeding behavior have been transformed repeatedly during frog evolution. Many of the independent lineages possess unique biomechanical mechanisms for protracting their tongues and unique neural mechanisms for coordinating feeding behavior. In frogs, there has been considerable evolution at the interface between reticular central pattern generators (CPGs) associated with feeding and sensory feedback circuits that modulate feeding motor output. In particular, the roles of hypoglossal and glossopharyngeal sensory feedback appear to have been relatively plastic in their evolution. Prey-type dependence of hypoglossal sensory feedback in Rana suggests that the interaction between descending visual control and sensory feedback also may be evolutionarily plastic. Comparative studies have found that motor systems sometimes evolve conservatively across morphological and behavioral transitions (i.e., the shoulder in birds) or, alternatively, they may be subject to considerably more evolutionary change than is reflected in morphological characteristics (i.e., feeding in cichlids). We hypothesize that the CPG circuits for feeding behavior in the reticular formation may evolve conservatively because they are highly integrated, multifunctional networks which cannot be optimized for one function without compromising others. In contrast, the interfaces between the CPG, sensory feedback and descending control should be less constrained. When changes in motor patterns occur during evolution, it is likely that sensory feedback or descending control may be involved.

Animals↗
PubMed1992

The nuclear serine/threonine protein kinase DNA-PK.

The DNA-activated protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase that phosphorylates DNA-binding proteins, including several transcription factors. DNA-PK is one of a very few enzymes known to be regulated through interaction with DNA that does not have DNA as a template or substrate. We suggest that DNA-PK may function in cell homeostasis, in part through the modulation of transcriptional activity.

Amino Acid Sequence↗