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C W Anderson

Publications and source records attributed to C W Anderson.

At least 37 records · Page 2Linked to original sources
PubMed1999

Properties of conditioned abducens nerve responses in a highly reduced in vitro brain stem preparation from the turtle.

Previous work suggested that the cerebellum and red nucleus are not necessary for the acquisition, extinction, and reacquistion of the in vitro classically conditioned abducens nerve response in the turtle. These findings are extended in the present study by obtaining conditioned responses (CRs) in preparations that received a partial ablation of the brain stem circuitry. In addition to removing all tissue rostral to and including the midbrain and cerebellum, a transection was made just caudal to the emergence of the IXth nerve. Such ablations result in a 4-mm-thick section of brain stem tissue that functionally eliminates the sustained component of the unconditioned response (UR) while leaving only a phasic component. We refer to this region of brain stem tissue caudal to the IXth nerve as the "caudal premotor blink region." Neural discharge was recorded from the abducens nerve following a single shock unconditioned stimulus (US) applied to the ipsilateral trigeminal nerve. When the US was paired with a conditioned stimulus (CS) applied to the posterior eighth, or auditory, nerve using a delay conditioning paradigm, a positive slope of CR acquisition was recorded in the abducens nerve, and CR extinction was recorded when the stimuli were alternated. Resumption of paired stimuli resulted in reacquisition. Quantitative analysis of the CRs in preparations in which the caudal premotor blink region had been removed and those with cerebellar/red nucleus lesions showed that both types of preparations had abnormally short latency CR onsets compared with preparations in which these regions were intact. Preparations with brain stem transections had significantly earlier CR offsets as more CRs terminated as short bursts when compared with intact or cerebellar lesioned preparations. These data suggest that a highly reduced in vitro brain stem preparation from the turtle can be classically conditioned. Furthermore, the caudal brain stem is not a site of acquisition in this reduced preparation, but it contributes to the sustained activity of both the UR and CR. Finally, the unusually short CR onset latencies following lesions to the cerebellum are not further exacerbated by removal of the caudal brain stem. These studies suggest that convergence of CS and US synaptic inputs onto the abducens nerve reflex circuitry may underlie acquisition in this reduced preparation, but that mechanisms that control learned CR timing arise from the cerebellorubral system.

Abducens Nerve↗
PubMed1998

DNA damage activates p53 through a phosphorylation-acetylation cascade.

Activation of p53-mediated transcription is a critical cellular response to DNA damage. p53 stability and site-specific DNA-binding activity and, therefore, transcriptional activity, are modulated by post-translational modifications including phosphorylation and acetylation. Here we show that p53 is acetylated in vitro at separate sites by two different histone acetyltransferases (HATs), the coactivators p300 and PCAF. p300 acetylates Lys-382 in the carboxy-terminal region of p53, whereas PCAF acetylates Lys-320 in the nuclear localization signal. Acetylations at either site enhance sequence-specific DNA binding. Using a polyclonal antisera specific for p53 that is phosphorylated or acetylated at specific residues, we show that Lys-382 of human p53 becomes acetylated and Ser-33 and Ser-37 become phosphorylated in vivo after exposing cells to UV light or ionizing radiation. In vitro, amino-terminal p53 peptides phosphorylated at Ser-33 and/or at Ser-37 differentially inhibited p53 acetylation by each HAT. These results suggest that DNA damage enhances p53 activity as a transcription factor in part through carboxy-terminal acetylation that, in turn, is directed by amino-terminal phosphorylation.

Acetylation↗
PubMed1998

Enhanced phosphorylation of p53 by ATM in response to DNA damage.

The ATM protein, encoded by the gene responsible for the human genetic disorder ataxia telangiectasia (A-T), regulates several cellular responses to DNA breaks. ATM shares a phosphoinositide 3-kinase-related domain with several proteins, some of them protein kinases. A wortmannin-sensitive protein kinase activity was associated with endogenous or recombinant ATM and was abolished by structural ATM mutations. In vitro substrates included the translation repressor PHAS-I and the p53 protein. ATM phosphorylated p53 in vitro on a single residue, serine-15, which is phosphorylated in vivo in response to DNA damage. This activity was markedly enhanced within minutes after treatment of cells with a radiomimetic drug; the total amount of ATM remained unchanged. Various damage-induced responses may be activated by enhancement of the protein kinase activity of ATM.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Distribution of hypoglossal motor neurons innervating the prehensile tongue of the African pig-nosed frog, Hemisus marmoratum.

Using retrograde neuronal tracers, a study of the distribution of hypoglossal motor neurons innervating the tongue musculature was performed in the African pig-nosed frog, Hemisus marmoratum. This species is a radically divergent anuran amphibian with a prehensile tongue that can be aimed in three dimensions relative to the head. The results illustrate a unique rostrocaudal distribution of the ventrolateral hypoglossal nucleus and an unusually large number of motor neurons within this cell group. During the evolution of the long, prehensile tongue of Hemisus, the motor neurons innervating the tongue have greatly increased in number and have become more caudally distributed in the brainstem and spinal cord compared to other anurans. These observations have implications for understanding neuronal reconfiguring of motoneurons for novel morphologies requiring new muscle activation patterns.

Animals↗
PubMed1998

The promoters for human DNA-PKcs (PRKDC) and MCM4: divergently transcribed genes located at chromosome 8 band q11.

A 30-kb genomic segment containing the promoter and first 9 exons of PRKDC, the gene encoding the catalytic subunit (DNA-PKcs) of the human DNA-activated protein kinase, DNA-PK, was isolated and partially sequenced. Sequence comparison with the NCBI nonredundant database revealed the locations of the first 13 exons of the upstream gene, MCM4. MCM4 is an essential component of a protein complex that prevents DNA from being replicated more than once per cell cycle. The MCM4 and DNA-PKcs promoters are in CpG islands separated by approximately 700 bp, and transcription from each initiates at multiple, closely spaced sites. Both promoters lack TATA boxes, and the MCM4 promoter also lacks an initiator (Inr) element but has an inverted CCAAT box. The DNA-PKcs promoter has an Inr-like sequence as well as a downstream MED-1 element. The two promoters appear to function independently, as sequences required for core promoter activity do not overlap, and sequences extending into the 5' region of each gene had little or no effect on transcription of the other gene, as shown in transient transfection assays. The arrangement of the PRKDC/MCM4 gene pair is similar to that of the ATM/E14(NPAT) gene pair. ATM, the product of the gene mutated in ataxia telangiectasia, and DNA-PKcs function in pathways that detect or repair DNA damage and are members of a family of large, serine/threonine kinases that are closely related to phosphatidylinositol 3 kinases.

Amino Acid Sequence↗
PubMed1998

Evidence for a photosensitive region in the caudal mesencephalon of the turtle brain.

Using an in vitro brainstem-cerebellum preparation from the turtle Chrysemys picta, burst discharge was recorded from the abducens nerve when light was directed on the brainstem. This burst discharge likely represents a neural correlate of the eye-blink reflex. Increasing the intensity of the light stimulus reduced the response latency from a duration of many seconds to approximately 1-2 s. No response was recorded when the light source was covered. The response was present when infrared light was blocked, and it could only be produced when the light spectra contained wavelengths below approximately 550 nm. Lesion experiments reveal that the photosensitive area is located caudal to the trochlear nerve and rostral to the trigeminal nerve. Single-unit microelectrode recordings demonstrate that this region is tonically active in the dark and that activity is suppressed by light. Pharmacological results show that the light response is blocked by application of compounds that act as serotonergic antagonists, and that antagonists of noradrenergic receptors (alpha or beta) either have no effect or their actions are variable. Taken together, these data suggest that an isthmo-optic-like area in the turtle brain is photosensitive to direct light and activates neural circuits that control eye movements.

Adrenergic Antagonists↗
PubMed1998

Multivariate autoregressive models for classification of spontaneous electroencephalographic signals during mental tasks.

This article explores the use of scalar and multivariate autoregressive (AR) models to extract features from the human electroencephalogram (EEG) with which mental tasks can be discriminated. This is part of a larger project to investigate the feasibility of using EEG to allow paralyzed persons to control a device such as a wheelchair. EEG signals from four subjects were recorded while they performed two mental tasks. Quarter-second windows of six-channel EEG were transformed into four different representations: scalar AR model coefficients, multivariate AR coefficients, eigenvalues of a correlation matrix, and the Karhunen-Loève transform of the multivariate AR coefficients. Feature vectors defined by these representations were classified with a standard, feedforward neural network trained via the error backpropagation algorithm. The four representations produced similar results, with the multivariate AR coefficients performing slightly better and more consistently with an average classification accuracy of 91.4% on novel, untrained, EEG signals.

Electroencephalography↗
PubMed1997

The cerebellum and red nucleus are not required for In vitro classical conditioning of the turtle abducens nerve response.

The role of the cerebellum during motor learning is a controversial issue. Many authors have suggested that the cerebellum and its connections with the red nucleus are essential for the acquisition of the conditioned eye blink reflex. Although there is little argument that the cerebellum is an important component to the generation of the conditioned response (CR), a number of studies have suggested that the cerebellum is not essential for conditioning. Using an in vitro model of the classically conditioned turtle abducens nerve response, we investigated the effect of cerebellar and red nucleus lesions on the acquisition, extinction, and reacquisition of CRs. Neural discharge was recorded from the abducens nerve after a single shock unconditioned stimulus (US) was applied to the ipsilateral trigeminal nerve. When the US was paired with a conditioned stimulus (CS) applied to the posterior eighth, or auditory, nerve, a positive slope of CR acquisition was recorded in the abducens nerve. After extinction stimuli in which the CS and US were alternated, the number of CRs decreased to near zero. When the CS and US were once again paired, reacquisition at a faster rate was recorded. The CRs showed unusual timing features compared with preparations in which the cerebellum was intact; they had significantly shorter latencies and showed burst-like responses. These data demonstrate that it is possible to classically condition this in vitro preparation in the absence of the cerebellum and red nucleus. However, the latencies of CRs were found to be dramatically altered in the cerebellar-lesioned preparations, suggesting that the cerebellum does play a role in the timing of the CR.

Abducens Nerve↗
PubMed1997

The functional anatomy and evolution of hypoglossal afferents in the leopard frog, Rana pipiens.

Previously, we suggested that afferents are present in the hypoglossal nerve of the leopard frog, Rana pipiens. The basis for this was behavioral data obtained after transection of the hypoglossal nerve. These afferents coordinate the timing of tongue protraction with mouth opening during feeding. The goal of the present study was to define anatomically these hypoglossal afferents in Rana pipiens. Retrograde tracing was performed using horseradish peroxidase, fluorescent dextran amines and neurobiotin. Data show that the cell bodies of hypoglossal afferents are located in the dorsal root ganglion of the third spinal nerve and enter the brainstem through its dorsal root. The afferents ascend in the dorsomedial funiculus and move laterally after they pass the obex. They project in the granular layer of the cerebellum and the medial reticular formation. The cervical afferents that travel in this pathway are known to carry proprioceptive and cutaneous sensory information. We hypothesize that the presence of afferents in the hypoglossal nerve is a derived characteristic of anurans, which has resulted from the re-routing of afferent fibers from the third spinal nerve into the hypoglossal nerve. The appearance of hypoglossal afferents coincides with the morphological acquisition of a highly protrusible tongue.

Animals↗
PubMed1997

Sites of UV-induced phosphorylation of the p34 subunit of replication protein A from HeLa cells.

Exposure of mammalian cells to UV radiation alters gene expression and cell cycle progression; some of these responses may ensure survival or serve as mutation-avoidance mechanisms, lessening the consequences of UV-induced DNA damage. We showed previously that UV irradiation increases phosphorylation of the p34 subunit of human replication protein A (RPA) and that this hyperphosphorylation correlated with loss of activity of the DNA replication complex. To characterize further the role of RPA hyperphosphorylation in the cellular response to UV irradiation and to determine which protein kinases might be involved, we identified by phosphopeptide analysis the sites phosphorylated in the p34 subunit of RPA (RPA-p34) from HeLa cells before and after exposure to 30 J/m2 UV light. In unirradiated HeLa cells, RPA-p34 is phosphorylated primarily at Ser-23 and Ser-29. At least four of the eight serines and one threonine in the N-terminal 33 residues of RPA-p34 can become phosphorylated after UV irradiation. Two of these sites (Ser-23 and Ser-29) are known to be sites phosphorylated by Cdc2 kinase; two others (Thr-21 and Ser-33) are consensus sites for the DNA-dependent protein kinase (DNA-PK); the fifth site (Ser-11, -12, or -13) does not correspond to the (Ser/Thr)-Gln DNA-PK consensus. All five can be phosphorylated in vitro by incubating purified RPA with purified DNA-PK. Two additional sites, probably Ser-4 and Ser-8, are phosphorylated in vivo after UV irradiation and in vitro by purified DNA-PK. The capacity of purified DNA-PK to phosphorylate many of these same sites on RPA-p34 in vitro implicates DNA-PK or a kinase with similar specificity in the UV-induced hyperphosphorylation of RPA in vivo.

Amino Acid Sequence↗
PubMed1997

Phosphorylation of serine 392 stabilizes the tetramer formation of tumor suppressor protein p53.

Tumor suppressor protein p53 is a tetrameric phosphoprotein that activates transcription from several cell cycle regulating genes in response to DNA damage. Tetramer formation is critical to p53's ability to activate transcription; however, posttranslational modifications and protein stabilization also contribute to p53's ability to activate transcription. To determine if phosphorylation affects tetramer formation, we synthesized phosphopeptides corresponding to residues 303-393 of human p53, which includes the domain responsible for tetramer formation. Phosphate was chemically incorporated at Ser315, Ser378, or Ser392 and also at both Ser315 and Ser392. Equilibrium ultracentrifugal analyses showed that phosphorylation at Ser392 increased the association constant for reversible tetramer formation nearly 10-fold. Phosphorylation of either Ser315 or Ser378 had little effect on tetramer formation, but phosphorylation of Ser315 largely reversed the effect of phosphorylation at Ser392. Analyses by calorimetry demonstrated that phosphorylation may influence subunit affinity (and, in turn, DNA binding) by an enthalpy-driven process, possibly between the C-terminal residues and the region immediately adjacent to Ser315. The Kd for the tetramer-monomer transition of the unphosphorylated p53 C-terminal domain was determined to be approximately 1-10 microM. Thus, in normal, undamaged cells p53 may be largely monomeric. Enhancement of tetramer formation through phosphorylation of Ser392, coupled with a DNA-damage-induced increase in its nuclear concentration, could provide a switch that activates p53 as a transcription factor in response to DNA damage.

Biopolymers↗
PubMed1997

Redox-mediated regulation of p21(waf1/cip1) expression involves a post-transcriptional mechanism and activation of the mitogen-activated protein kinase pathway.

p21(waf1/cip1) gene expression is induced by DNA damage in cells with wild-type p53 and contributes to the arrest of cell growth. It was demonstrated that under many experimental conditions, including oxidative stress, p21(waf1/cip1) expression can be induced through p53-independent pathways. Since most of these experimental conditions induce the phosphorylation of mitogen-activated protein kinase (MAPK) and thus its activation, we evaluated p21(waf1/cip1) mRNA levels in cells exposed to an oxidative stress, induced by diethylmaleate (Et2Mal), and in which the MAPK pathway was blocked. The expression of a dominant-negative mutant of MEK, the MAPK kinase that phosphorylates and activates MAPK, and of a dominant-negative [Asn17]Ras mutant prevented the Et2Mal-induced accumulation of p21(waf1/cip1) mRNA. Similarly, the expression of MEK- and of [Asn17]Ras mutants decreased the 12-O-tetradecanoyl-phorbol 13-acetate (TPA)-mediated p21(waf1/cip1) induction. Furthermore, TPA-induced and serum-induced p21(waf1/cip1) mRNA accumulation was blocked by pretreating the cells with the antioxidant compound N-acetylcysteine, suggesting that oxidative stress is involved in these responses. p21(waf1/cip1) mRNA levels reached a maximum within 2 h of adding Et2Mal or TPA; however, the rate of transcription from a p21(waf1/cip1)-promoter construct did not increase during this period. In contrast, cells treated with actinomycin D show an increase of p21(waf1/cip1) mRNA stability after Et2Mal treatment. This result suggests that the increase in p21(waf1/cip1) mRNA at early times results from post-transcriptional regulatory events. Longer exposure to TPA may activate p21(waf1/cip1) gene transcription through an Sp1-dependent mechanism, while Et2Mal treatment gradually inhibits p21(waf1/cip1) gene transcription through oxidative changes that affect Sp1 binding to DNA.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

Effect of phosphorylation on tetramerization of the tumor suppressor protein p53.

Human tumor suppressor protein p53 is a 393-amino acid phosphoprotein that enhances transcription in response to DNA damage from several genes that regulate cell cycle progression. The tetrameric state of p53 is critical to wild-type function; the p53 tetramerization element is located in the C-terminal region of the protein. This region is phosphorylated at several evolutionarily conserved serines, suggesting that phosphorylation may be an important regulator of p53 function. In order to determine the effect of phosphorylation on tetramer formation, we synthesized phosphopeptides corresponding to p53(Ser303-Asp393) with phosphate incorporated at Ser315, Ser378, or Ser392, and at both Ser315 and Ser392. Equilibrium ultracentrifugation analysis showed that phosphorylation at Ser392 increased the association constant for tetramer formation nearly ten-fold. By itself, phosphorylation at Ser315 or Ser378 had little effect on tetramer formation, but Ser315 largely reversed the effect of phosphorylation at Ser392. Analysis by calorimetry suggests that phosphorylation may influence subunit affinity by an enthalpy driven process.

Amino Acid Sequence↗
PubMed1997

Prolonged p53 protein accumulation in trichothiodystrophy fibroblasts dependent on unrepaired pyrimidine dimers on the transcribed strands of cellular genes.

Trichothiodistrophy (TTD), xeroderma pigmentosum (XP), and Cockayne's syndrome (CS) are three distinct human diseases with sensitivity to ultraviolet (UV) radiation affected by mutations in genes involved in nucleotide excision repair (NER). Among the many responses of human cells to UV irradiation, both nuclear accumulation of p53, a tumor suppressor protein, and alterations in cell-cycle checkpoints play crucial roles. The purpose of this study was to define the signals transmitted after UV-C-induced DNA damage, which activates p53 accumulation in TTD/XP-D fibroblasts, and compare this with XP-D cell lines that carry different mutations in the same gene, XPD. Our results showed that p53 was rapidly induced in the nuclei of TTD/XP-D and XP-D fibroblasts in a dose-dependent manner after UV-C irradiation, as seen in XP-A and CS-A fibroblasts, much lower doses being required for the protein accumulation than in normal human fibroblasts, XP variant cells, and XP-C cells. The kinetics of accumulation of p53 and two effector proteins involved in cell-cycle arrest, WAF1 and GADD45, were also directly related to the repair potential of the cells, as in normal human fibroblasts their levels declined after 24 h, the time required for repair of UV-induced lesions, whereas NER-deficient TTD/XP-D cells showed p53, WAF1, and GADD45 accumulation for over 72 h after irradiation. Our results indicate that p53 accumulation followed by transcriptional activation of genes implicated in growth arrest is triggered in TTD/XP-D cells by the persistence of cyclobutane pyrimidine dimers, which are known to block transcription, on the transcribed strands of active genes.

Adolescent↗
PubMed1996

Alternate splice-site utilization in the gene for the catalytic subunit of the DNA-activated protein kinase, DNA-PKcs.

Analysis of polyA-selected RNAs and cDNA clones from several human cell lines revealed the presence of a 93-bp exon in the PI kinase homology region of DNA-PKcs that was not present in the cDNA sequence derived from HeLa cell cDNA clones. RT-PCR showed that most DNA-PKcs mRNAs in human cells have this exon. Thus, the nascent DNA-PKcs polypeptide is composed of 4127 aa and has a predicted mol. wt. of 469021 (470 kDa).

Alternative Splicing↗