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Biomedical subjects

C W Anderson

Publications and source records attributed to C W Anderson.

At least 91 records · Page 5Linked to original sources
PubMed1992

Concurrent benign teratoid medulloepithelioma and pineoblastoma.

Teratoid medulloepithelioma of the ciliary body is a rare tumor usually occurring in young children. Primary tumors of the pineal gland are also quite rare and have a similar age distribution. We report the case of a 2-year-old girl who had a leukocoria of the right eye. Cataract surgery was performed; however, the patient developed a total retinal detachment refractory to treatment. She eventually developed early changes of phthisis bulbi and underwent an evisceration. Histopathologic examination revealed a benign teratoid medulloepithelioma containing islands of hyaline cartilage and rhabdomyoblasts. During the course of her evaluation, a pineoblastoma was discovered and surgically excised. While the association of pineal tumors with retinoblastomas has been well established, no such association has been reported for medulloepitheliomas. To our knowledge, this is the first recorded case of a benign teratoid medulloepithelioma occurring concurrently with a pineoblastoma.

Brain Neoplasms↗
PubMed1991

The protein-tyrosine kinase substrate, calpactin I heavy chain (p36), is part of the primer recognition protein complex that interacts with DNA polymerase alpha.

Primer recognition proteins (PRP) stimulate the activity of DNA polymerase alpha on DNA substrates with long single-stranded template containing few primers. Purified PRP from HeLa cells and human placenta are composed of two subunits of 36,000 (PRP 1) and 41,000 (PRP 2) daltons. By amino acid sequence homology, we have identified PRP 2 as the glycolytic enzyme 3-phosphoglycerate kinase. Here we present data that establishes PRP 1 to be the protein-tyrosine kinase substrate, calpactin I heavy chain. Amino acid sequence analysis of six tryptic peptides of PRP 1 followed by homology search in a protein sequence data base revealed 100% identity of all six peptides with the deduced amino acid sequence of human calpactin I heavy chain. The activities of PRP and calpactin I coelute on gel filtration columns, and a high correlation of PRP and calpactin I activities was seen at different stages of purification. A rabbit polyclonal anti-chicken calpactin I antibody was shown to cross-react with PRP 1 polypeptide at various stages of PRP purification, and the homogeneous preparation of PRP exhibits 3-phosphoglycerate kinase (PRP 2) and calpactin I (PRP 1) activities. PRP activity is neutralized by a mouse monoclonal anti-calpactin II antibody although having no effect on the polymerase alpha activity itself. Calpactin II has a 50% amino acid sequence homology with calpactin I. However, PRP 1 is not calpactin II as shown by lack of cross-reaction to a monoclonal anti-calpactin II antibody on Western blots. Calpactin I and 3-phosphoglycerate kinase, purified independently, cannot be efficiently reconstituted into the PRP complex, indicating that their association in the PRP complex involves specific protein-protein interactions that remain to be elucidated. The biochemical and immunological data presented here revealing the identity of PRP 1 as calpactin I provide evidence for one physiological role of calpactin I in the cell.

Amino Acid Sequence↗
PubMed1991

The retinoblastoma protein is phosphorylated on multiple sites by human cdc2.

The retinoblastoma gene product (pRB) is a nuclear phosphoprotein that is thought to play a key role in the negative regulation of cellular proliferation. pRB is phosphorylated in a cell cycle dependent manner, and studies in both actively dividing and differentiated cells suggest that this modification may be essential for cells to progress through the cell cycle. Using tryptic phosphopeptide mapping we have shown that pRB is phosphorylated on multiple serine and threonine residues in vivo and that many of these phosphorylation events can be mimicked in vitro using purified p34cdc2. Using synthetic peptides corresponding to potential cdc2 phosphorylation sites, we have developed a strategy which has allowed the identification of five sites. S249, T252, T373, S807 and S811 are phosphorylated in vivo, and in each case these sites correspond closely to the consensus sequence for phosphorylation by p34cdc2. This and the observation that pRB forms a specific complex with p34cdc2 in vivo suggests that p34cdc2 or a p34cdc2-related protein is a major pRB kinase.

Amino Acid Sequence↗
PubMed1991

Catalysis of poliovirus VP0 maturation cleavage is not mediated by serine 10 of VP2.

The maturation of the poliovirus capsid occurs as the result of a single unexplained proteolytic event during which 58 to 59 copies of the 60 VP0 capsid protein precursors are cleaved. An autocatalytic mechanism for cleavage of VP0 to VP4 and VP2 was proposed by Arnold et al. (E. Arnold, M. Luo, G. Vriend, M. G. Rossman, A. C. Palmenberg, G. D. Parks, M. J. Nicklin, and E. Wimmer, Proc. Natl. Acad. Sci. USA 84:21-25, 1987) in which serine 10 of VP2 is activated by virion RNA to catalyze VP4-VP2 processing. The hypothesis rests on the observation that a hydrogen bond was observed between serine 10 of VP2 (S2010) and the carboxyl terminus of VP4 in three mature picornaviral atomic structures: rhinovirus 14, mengovirus, and poliovirus type 1 (Mahoney). We constructed mutant viruses with cysteine (S2010C) or alanine (S2010A) replacing serine 10 of VP2; these exhibited normal proteolytic processing of VP0. While our results do not exclude an autocatalytic mechanism for the maturation cleavage, they do eliminate the conserved S2010 residue as the catalytic amino acid.

Amino Acid Sequence↗
PubMed1991

The human DNA-activated protein kinase phosphorylates simian virus 40 T antigen at amino- and carboxy-terminal sites.

Protein phosphorylation modulates the functions of simian virus 40 large T antigen (TAg) in productive and transforming infections. We recently described a DNA-activated protein kinase (DNA-PK) that efficiently phosphorylates TAg and several other nuclear, DNA-binding proteins in vitro (S.P. Lees-Miller, Y.-R. Chen, and C. W. Anderson, Mol. Cell. Biol. 10:6472-6481, 1990). In this report, we show by direct amino acid sequence analysis that DNA-PK phosphorylates TAg strongly at Ser-677, a residue known to be important for TAg interaction with origin site I and for transformation. We propose that DNA-PK may modulate the role of TAg in repressing early viral transcription and cell transformation, but a role for DNA-PK in regulating simian virus 40 DNA synthesis is not excluded. DNA-PK also phosphorylates Ser-665, and Ser-667, and one or more serines between amino acids 110 and 131. At least six serines, Ser-111, Ser-112, Ser-120, Ser-665, Ser-667, and Ser-677, are phosphorylated in TAg purified from baculovirus vector-infected insect cells.

Amino Acid Sequence↗
PubMed1991

The DNA-activated protein kinase, DNA-PK: a potential coordinator of nuclear events.

DNA-PK is a moderately abundant serine/threonine protein kinase found in the nucleus of a wide range of eukaryotic cells. It is one of the few known cellular enzymes whose activity is regulated directly by DNA. Many DNA binding proteins, including a number of transcription factors, are substrates for DNA-PK in vitro. We suggest that this kinase may coordinate signal transduction pathways and nuclear events, including transcription, in response to changes in DNA or chromatin state.

Amino Acid Sequence↗
PubMed1990

Suramin affects DNA synthesis in HeLa cells by inhibition of DNA polymerases.

Suramin, a polysulfonated naphthylurea widely used in the treatment of trypanosomiasis and onchocerciasis, is currently being investigated as an antitumor agent for the treatment of advanced cancer. Suramin exerts a wide variety of biological effects. We have shown that suramin inhibits cell proliferation and DNA synthesis in cultured HeLa cells. The replication in vitro of SV40 DNA is completely abolished by 40 microM suramin. The inhibition of DNA replication is due to inhibition of DNA polymerases alpha and delta, the replicative enzymes in eukaryotic cells. DNA polymerase alpha is sensitive to lower concentrations of suramin [concentration to achieve 50% inhibition (IC50) of 8 microM] than is DNA polymerase delta (IC50 36 microM), whereas DNA polymerase beta is relatively insensitive to the drug (IC50 of 90 microM). Suramin inhibits other replicative DNA polymerases such as Escherichia coli polymerase I (Klenow fragment) and Thermus aquaticus polymerase. Suramin is noncompetitive with both substrate deoxyribonucleotides and template-primers with respect to DNA polymerase inhibition. Much lower concentrations (8-30 microM) of the drug are required for 50% inhibition of DNA polymerases than for 50% inhibition of other enzymes such as protein kinase C and reverse transcriptase. These results show an important biological effect of this drug and indicate the need for more studies before its clinical use as an antitumor agent.

DNA Polymerase II↗
PubMed1990

Drosophila proliferating cell nuclear antigen. Structural and functional homology with its mammalian counterpart.

A protein with an apparent mass of 36 kDa was purified from Drosophila melanogaster embryos using a protocol developed for the purification of proliferating cell nuclear antigen (PCNA) from human 293 cells. The Drosophila protein comigrated with human PCNA on one-dimensional sodium dodecyl sulfate-polyacrylamide gels and cross-reacted with monoclonal anti-rabbit PCNA antibodies. NH2-terminal amino acid sequence analysis revealed that the putative Drosophila PCNA was highly homologous to human PCNA. Of the first 22 amino acids, 16 were identical, and 4 of the remaining 6 were changed conservatively. Results of total amino acid analysis were also consistent with a high degree of similarity between Drosophila PCNA and human PCNA. Functional analysis using the reconstituted simian virus 40 in vitro DNA replication system demonstrated that Drosophila PCNA could substitute, albeit with reduced efficiency, for human PCNA in stimulating simian virus 40 DNA synthesis. Affinity-purified anti-Drosophila PCNA antibodies cross-reacted with human PCNA and were able to recognize specifically Drosophila PCNA both on crude homogenate immunoblots and by indirect immunofluorescence analysis of proliferating cells in larval tissues in situ. These antibodies thus promise to be useful probes for the study of cell proliferation in this rapidly developing organism.

Amino Acid Sequence↗
PubMed1990

The proteinase polypeptide of adenovirus serotype 2 virions.

The Ad2 proteinase, which is thought to be encoded by a 23-kDa open reading frame located at the end of the L3 family of late mRNAs, is expressed poorly even late after infection. To obtain sufficient proteinase for biochemical characterization, a DNA fragment containing the 23-kDa open reading frame was cloned into plasmids that permit efficient expression in Escherichia coli. Polyclonal antiserum specific for the Ad2 proteinase was produced by immunizing rabbits with a fusion protein that included the entire proteinase open reading frame, and this antiserum was used to show that the product of the 23-kDa reading frame is assembled into virions. Bacterial products corresponding to the complete 204 amino acid proteinase reading frame, to a 9 amino acid proteinase deletion, and to a proteinase fusion protein of 227 amino acids were used to determine the size of the proteinase polypeptide in Ad2 virions and in infected HeLa cell extracts. A single proteinase polypeptide that migrated during SDS-polyacrylamide gel electrophoresis with the 204 amino acid recombinant proteinase was detected in wild-type and H2ts1 virions, and in infected cell extracts. Immunoblot titrations showed that a wild-type Ad2 virus particle contains about 10 proteinase polypeptides; an H2ts1 virion has approximately fivefold less proteinase. In virions, the proteinase was associated primarily with the virus core. The 204 amino acid proteinase produced in E. coli permitted cleavage of the major core protein precursor, P-VII, to mature, authentic VII, but the proteinase deletion lacking 9 amino acids from near the amino-terminus was inactive. These results are inconsistent with autocatalytic processing of the Ad2 proteinase as was reported by Chatterjee and Flint (1987, Proc. Natl. Acad. Sci. USA 84, 714-718).

Adenoviruses, Human↗
PubMed1990

Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen.

HeLa cells contain a serine/threonine protein kinase (DNA-PK) that is strongly activated in vitro by low concentrations of double-stranded DNA (dsDNA). Activation was specific for dsDNA; both natural DNAs and synthetic oligonucleotides functioned as kinase activators. The fact that DNA-PK activity was rapidly inhibited by incubation with dsDNA and ATP suggests that DNA-PK activity also may be regulated by autophosphorylation. During gel filtration, DNA-PK activity behaved as a 350-kDa protein, and highly purified DNA-PK contained a dsDNA-binding, 350-kDa polypeptide that was phosphorylated in a dsDNA-dependent manner. We conclude that this 350-kDa polypeptide is likely to be DNA-PK. Previously we showed that the dsDNA-activated kinase phosphorylates two threonines at the N terminus of hsp90 alpha (S. P. Lees-Miller and C. W. Anderson, J. Biol. Chem. 264:17275-17280, 1989). Here we show that DNA-PK also phosphorylates the simian virus 40 large tumor antigen, the mouse tumor-suppressor protein p53, the human Ku autoantigen, and two unidentified HeLa DNA-associated polypeptides of 52 and 110 kDa. Identification of these and other newly identified DNA-binding substrates suggest that the dsDNA-activated kinase may regulate transcription, DNA replication, or cell growth.

Amino Acid Sequence↗
PubMed1989

The human double-stranded DNA-activated protein kinase phosphorylates the 90-kDa heat-shock protein, hsp90 alpha at two NH2-terminal threonine residues.

The 90-kDa heat-shock protein, hsp90, is an abundant cytoplasmic protein that can be phosphorylated in vitro by a double-stranded (ds) DNA-activated protein kinase found in cells from several species. Here we show that the dsDNA-activated protein kinase from human HeLa cells phosphorylates 2 threonine residues in the sequence PEETQTQDQPME at the amino terminus of human hsp90 alpha. Hsp90 beta, which is 97% identical to hsp90 alpha but lacks both amino-terminal threonines, is not phosphorylated by the dsDNA-activated protein kinase. Mouse hsp86 and rabbit hsp90 alpha are homologous to human hsp90 alpha; both heterologous proteins are phosphorylated at the same amino-terminal threonines by the human dsDNA-activated protein kinase.

Amino Acid Sequence↗
PubMed1989

Two human 90-kDa heat shock proteins are phosphorylated in vivo at conserved serines that are phosphorylated in vitro by casein kinase II.

Amino-terminal protein sequence analysis revealed that exponentially growing human HeLa cells at 37 degrees C express two closely related 90-kDa "heat shock" proteins (hsp 90) in nearly equal amounts. Both hsp 90s begin with proline; the initial methionine residue is removed. The alpha protein contains a 9-amino acid segment, TQTQDQPME, from residues 4 to 12, that is replaced by a 4-amino acid segment, VHHG, in the beta form. The purified hsp 90 mixture contains 2 mol of phosphate/mol of polypeptide. Both hsp 90 proteins are phosphorylated at two homologous sites. For the alpha protein, these sites correspond to serine 231 and serine 263. A 5-amino acid segment, ESEDK, between the two phosphorylation sites is absent from the beta protein. The sequence between phosphorylation sites of both hsp 90s is predicted to have alpha helical structure. Dephosphorylated hsp 90 is phosphorylated at both sites by casein kinase II from HeLa cells, calf thymus, or rabbit reticulocytes; no other hsp 90 residues were phosphorylated by casein kinase II in vitro.

Amino Acid Sequence↗
PubMed1989

Characterization of the adenovirus 2 virion protein, mu.

Adenovirus 2 virions contain a small, highly basic protein known as mu (mu). Partial sequence analysis of mu labeled with radioactive amino acids showed that it is derived from an 11-kDa virion precursor protein, L2-79R. Amino acid analysis, direct microsequence analysis, time-of-flight mass spectrometer analysis, and chemical synthesis demonstrated that mu is the unmodified, 19 amino acid peptide obtained from the 79-residue precursor by adenovirus-encoded proteinase-mediated cleavage after glycine31 and glycine50. Mu bound tightly to DNA and was located in the virion core. In vitro, mu could precipitate DNA fragments, suggesting that it may have a role in viral chromosome condensation.

Adenoviruses, Human↗
PubMed1989

Gastric ulcers in horses: a comparison of endoscopic findings in horses with and without clinical signs.

Gastroendoscopic examinations were performed on 187 horses, ranging from one to 24 years. Eighty-seven horses had clinical problems including chronic, recurrent colic for seven or more days (25), one or more episodes of colic within the previous seven days (13), or acute colic (10), diminished appetite (53), poor bodily condition (40), and/or chronic diarrhoea (9). One hundred horses that had no signs of gastrointestinal problems were examined as part of a gastroendoscopic survey. Lesions observed in the squamous fundus, squamous mucosa adjacent to the margo plicatus along the greater curvature, glandular fundus, and the squamous mucosa along the lesser curvature were scored on a scale of 0-4, with 0 representing no lesions and 4 representing the most severe lesions. The mean endoscopic scores for the squamous fundus, margo plicatus and lesser curvature were significantly greater (P < 0.001) in horses with clinical signs than those without signs. This was because of the greater number of horses with lesions in the symptomatic group (80/87) compared to those without signs (52/100), and the greater severity of lesions in the horses with clinical signs. Of the horses, 74 were in race training. There was a significantly (P < 0.01) greater prevalence and severity of lesions at all sites except the glandular fundus in horses in training compared to those not in training, and in the horses in training with clinical signs (n = 37) compared to those in training without clinical signs (n = 37).

Animals↗
PubMed1988

A non-AUG translational initiation in c-myc exon 1 generates an N-terminally distinct protein whose synthesis is disrupted in Burkitt's lymphomas.

The c-myc gene comprises three exons with a single large AUG-initiated open reading frame extending from exon 2 through exon 3. Exon 1 lacks any AUG codons. Cells from a wide range of species produce two c-myc proteins that, while highly related, do not appear to arise from posttranslational interconversion. To understand the origin of the two proteins, we mapped them and analyzed the in vitro protein-coding capacity of c-myc cDNAs. Our findings show that the two proteins are derived from alternative translational initiations at the exon 2 AUG and at a non-AUG codon near the 3' end of exon 1, resulting in the production of proteins with distinct N termini. In Burkitt's lymphomas, the removal or specific mutation of exon 1 in c-myc translocations correlates with suppression of synthesis of the larger protein, and thus may contribute to the oncogenic activation of c-myc.

Animals↗
PubMed1988

Abnormal expression of a late gene family L1 protein in monkey cells abortively infected with adenovirus type 2.

The drastically reduced virus yields obtained from monkey cells abortively infected with adenovirus 2 (Ad2) have been attributed primarily to a severe decrease in the accumulation of the virion protein fiber (IV), a product of the most pomoter distal late gene family, L5. Here we report that the accumulation of virion protein IIIa, a product of the proximal late gene family, L1, is also severely depressed. In contrast, the i-leader protein LO-13.6K and L1 protein(s) 52K/55K are expressed with the same time course and in equal amounts in monkey cells abortively infected by Ad2 or productively infected by the Ad2-simian virus 40 (SV40) hybrid Ad2+ND1 or by the host range mutant Ad2+ND3 hr602. L1-52K/55K is phosphorylated in abortively infected CV-1 or CV-C monkey cells as well as in productively infected human and monkey cells. As with fiber expression, the failure to produce IIIa appears to be due partly to reduced or delayed IIIa mRNA accumulation. The small amount of IIIa protein that is synthesized in monkey cells is stable. Since the accumulation of both IIIa and fiber protein is deficient, the mechanism of abortive infection cannot be attributed solely to the absence of the auxiliary fiber leader sequences (1).

Adenoviruses, Human↗
PubMed1988

Interaction of human adenovirus serotype 2 with human lymphoid cells.

Human adenoviruses (e.g., Ad2, Ad5) establish chronic infections in human lymphoid-derived cell lines, including Raji and Jijoye (R.E. Wallace, 1969, Proc. Soc. Exp. Biol. Med. 130, 702-710; N. Faucon, G. Ogier, and Y. Chardonnet, 1982, J. Natl. Cancer Inst. 69, 1215-1220); however, the mechanisms by which chronic infections are established and maintained are not understood. When Raji or MOLT-3 cell cultures were infected with Ad2 at high multiplicity, these cell lines continued to grow exponentially and produced only small amounts of infectious virus. Virus-specific antigens, including the DNA-binding protein and hexon, were expressed in only 5% of the Ad2-inoculated cultures. All Raji and MOLT-3 cells were found to have adenovirus receptors, but the Ad2 virions that adsorbed to most Raji cells were sequestered in caps, suggesting that most cells fail to internalize adsorbed Ad2. Cell synchronization experiments showed a correlation between the proportion of cells that became productively infected and the proportion of cells in mitosis at the time of infection. In contrast, primary blood lymphocytes had few, if any, Ad2 receptors and were not productively infected by Ad2.

Adenoviruses, Human↗