Search PubMed⌕ Search

Biomedical subjects

C A Carter

Publications and source records attributed to C A Carter.

At least 55 records · Page 3Linked to original sources
PubMed1992

Assembly of recombinant human immunodeficiency virus type 1 capsid protein in vitro.

The capsid protein (CA) (p24) of human immunodeficiency virus (HIV) type 1 expressed in Escherichia coli and purified to greater than 90% homogeneity was used to examine assembly in vitro and to probe the nature of interactions involved in the formation of capsid structures. The protein was detected in dimeric and oligomeric forms as indicated by molecular size measurements by gel filtration column chromatography, sedimentation through sucrose, and nondenaturing gel electrophoresis. Chemical cross-linking of CA molecules was observed with several homobifunctional reagents. Oligomer size was dependent on cross-linker concentration and exhibited a nonrandom pattern in which dimers and tetramers were more abundant than trimers and pentamers. Oligomers as large as dodecamers were detected in native polyacrylamide gels. These were stable in solutions of high ionic strength or in the presence of nonionic detergent, indicating that strong interactions were involved in oligomer stabilization. Limited tryptic digestion converted the putative dodecamers to octamers, suggesting that a region involved in CA protein multimerization was exposed in the structure. This region was mapped to the central portion of the protein. The recombinant CA proteins assembled in vitro into long rodlike structures and were disassembled into small irregular spheres by alterations in ionic strength and pH. The observation that assembly and disassembly of purified HIV type 1 CA protein can be induced in vitro suggests an approach for identifying possible control mechanisms involved in HIV viral core assembly.

Capsid↗
PubMed1992

Effects of the SV40 large T antigen and EJ ras oncogene on fibronectin localization in human endometrial cells as viewed by confocal laser scanning microscopy.

We utilized confocal laser scanning microscopy to examine the localization of fibronectin deposition in cultures of human endometrial stromal cells. We found that fibronectin in normal human endometrial stromal cell cultures was both intracellular, occurring in rough endoplasmic reticulum and in perinuclear regions, and extracellular, occurring diffusely over the entire cell surface. Endometrial stromal cells were transfected with a plasmid containing an origin-defective Simian Virus 40 (SV40) which codes for a temperature-sensitive large T antigen. When these cells were placed under temperature-restrictive conditions for large T-antigen function, they exhibited staining patterns similar to normal endometrial cells. Fibronectin deposition in cultures of partially or fully transformed endometrial cells was not intracellular as in normal cells, but was localized primarily between cells. Cells expressing the SV40 large T antigen deposited fibronectin mainly in parallel clumps between cells. Cells expressing both the SV40 large T antigen and the EJ ras oncogene, at high cell density, displayed networks of fibronectin arranged in matrix-like patterns between cells. The malignant cell line examined, sarcoma cells, also exhibited fibronectin networks between cells. Cell density affected fibronectin deposition in endometrial stromal cells expressing the EJ ras oncogene. At low density, cells expressing the SV40 large T antigen and the EJ ras oncogene displayed diffuse fibronectin patterns and, at high density, these cells formed colonies with networks of fibronectin between cells.

Antigens, Polyomavirus Transforming↗
PubMed1992

The formation and enzymatic repair of DNA modifications caused by the haloethylnitrosoureas and related compounds.

The haloethylnitrosoureas are both useful antitumor agents and known carcinogens. These biological activities are believed to be associated with DNA modification, and some biologically significant lesions have been identified in DNA exposed to these agents. At the same time, DNA repair is a cause of resistance to treatment by these agents, and may also serve as protection against their carcinogenic effects.

Animals↗
PubMed1991

Release of 7-alkylguanines from N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA by 3-methyladenine DNA glycosylase II.

Purified bacterial 3-methyladenine DNA glycosylase II releases four 7-alkylguanines from [3H]N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA: 7-(2-hydroxyethyl)guanine,1,2-bis(7-guanyl)ethane, 7-(2-chloroethyl)guanine, and 7-(2-ethoxyethyl)guanine. 7-(2-Ethoxyethyl)guanine, a new compound, is formed as a result of an interaction with ethanol, a common solvent for the 2-haloethylnitrosoureas. Of the four 7-alkylguanines which are released from [3H]N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-modified DNA, 7-(2-hydroxyethyl)guanine is released at a rate very much slower than the other three. As shown by a study of the spontaneous decomposition of the corresponding 7-alkyl-deoxyguanines, differences in chemical stability do not appear to explain the slow release of 7-(2-hydroxyethyl)guanine. In view of previous results showing a difference in the distribution of alkylation products between sensitive and resistant glial cell lines, the broad specificity of this enzyme suggests that glycosylase activity could play a role in cellular resistance to 2-haloethylnitrosoureas.

Alkylation↗
PubMed1991

Cell behavior during early development in the South American annual fishes of the genus Cynolebias.

Living embryos of three species of South American annual fishes, Cynolebias constanciae, C. nigripinnis, and C. whitei, were observed from fertilization through the 10-somite stage. A description of normal stages of development applicable to all three species of Cynolebias is presented. Cleavage (stages 1-10) is meroblastic and produces a typical teleost blastoderm. Following cleavage (stages 11-13) blastomeres segregate into two populations, viz., 1) a population of deep blastomeres that will disperse as single motile cells, and 2) a hemispherical shell of outer blastomeres that flattens to form an enveloping cell layer (EVL). When epiboly of the EVL and the yolk syncytial layer (YSL) commences (stage 14), deep blastomeres clump together as a consolidation mass and then migrate outward as single cells on the YSL. When epiboly is concluded (stage 19), deep blastomeres have completely dispersed. If diapause does not intervene, the dispersed phase lasts only a few days. Subsequently, the dispersed cells come together to form a definitive aggregate (stage 27). Embryogenesis within the reaggregated mass of previously dispersed cells produces a typical teleost embryo. Early development in Cynolebias resembles that of other South American annual fishes, such as Austrofundulus, in that a phase of deep blastomere dispersion and reaggregation spatially and temporally separates epiboly from embryogenesis. Several features of development markedly differ from Austrofundulus. There are far fewer (250 vs. 2,500) deep blastomeres. Deep cells of Cynolebias are flattened rhomboids with filipodial extensions in contrast to the amoeboid cells of Austrofundulus. Blastomeres of dispersion and reaggregation stages in Cynolebias send out numerous cell surface extensions onto the YSL and in contact with one another, and often line up in rows as do some African annual fishes, e.g., Nothobranchius. During Dispersion II (stage 21), Reaggregation I (stage 22), and Reaggregation II (stage 23), deep cells move in an oriented pattern with respective mean velocities of 3.48 +/- 0.91, 1.28 +/- 0.46, and 1.31 +/- 0.31 microns/minute. Cells move toward a granular mass of unknown composition, located at the YSL-yolk interface in the lower hemisphere of the egg. This mass appears to coincide with the site of cell reaggregation.

Animals↗
PubMed1991

Effects of isomeric 2-(arylmethylamino)-1,3-propanediols (AMAPs) and clinically established agents on macromolecular synthesis in P388 and MCF-7 cells.

The in vitro effects of the 2-(arylmethylamino)-1,3-propanediols (AMAPs) on macromolecular synthesis have been examined using the murine leukemia, P388, and the human mammary adenocarcinoma, MCF-7, under conditions of short-term drug exposure. AMAPs that were observed to inhibit macromolecular synthesis produced nearly equipotent inhibition of DNA and RNA synthesis. Equivalent inhibition of protein synthesis generally required significantly greater concentrations of AMAP. There is a general correlation between inhibition of polynucleotide synthesis and in vivo antitumor activity. The effects of four clinical candidate AMAPs (crisnatol, 773U82, 502U83, and 7U85) on macromolecular synthesis were further compared with those of actinomycin D, doxorubicin, mitoxantrone, etoposide, amsacrine, and cisplatin in MCF-7 cells. The pattern of AMAP action was most similar to that observed for doxorubicin and mitoxantrone. Finally, the effects of these four AMAPs on the size, specific activity, and rate of incorporation of [3H]-dTTP into DNA of MCF-7 cells synchronized by pretreatment with hydroxyurea was determined. It was found that DNA synthesis was inhibited by AMAPs independent of inhibition of the uptake, phosphorylation, or retention of the metabolic precursors. These results support the theory that antitumor AMAPs interfere with the normal functioning of enzymes, such as topoisomerase II or DNA and RNA polymerases, which interact with DNA.

Animals↗
PubMed1991

Release of N2,3-ethanoguanine from haloethylnitrosourea-treated DNA by Escherichia coli 3-methyladenine DNA glycosylase II.

3-Methyladenine DNA glycosylase II (Gly II), purified from Escherichia coli cells which carry the plasmid PYN1000, has been tested for its ability to release N2,3-ethanoguanine from DNA modified by the antitumor agent N-[2-chloroethyl-1,2-14C]-N'-cyclohexyl-N-nitrosourea ([14C]CCNU). Gly II has been shown to release N2,3-ethanoguanine in a protein- and time-dependent manner at a rate that exceeds the rate at which this enzyme releases other alkylated bases from [14C]CCNU-modified DNA. This finding widens the known substrate specificity for Gly II to include a modified base which bears an exocyclic ring structure, a class of modifications caused by a variety of chemical carcinogens.

Bacterial Proteins↗
PubMed1991

Fluorescent laser scanning microscopy of F-actin disruption in human endometrial stromal cells expressing the SV40 large T antigen and the EJ ras oncogene.

To attempt to understand the effects of the SV40 large T antigen and an activated EJ ras oncogene on F-actin organization, we compared normal human endometrial stromal cells (HESC; proliferating, short life span) to cells transfected with the SV40 large T antigen either alone or in combination with the EJ ras oncogene. Normal HESC displayed numerous bundles of actin filaments (stress fibers) evenly distributed throughout the cell. In HESC transfected with a plasmid containing the gene for a temperature-sensitive SV40 large T antigen, stress fibers were disrupted and the remaining F-actin was also disrupted and clumped near the plasma membrane. Cells expressing both the SV40 large T antigen and the EJ ras oncogene sometimes appeared rounded, with stress fibers organized mainly near the cell periphery. Under restrictive temperature conditions for the function of the SV40 large T antigen, cells with or without the EJ ras oncogene reorganize actin stress fibers to resemble those of normal HESC. Therefore, the EJ ras oncogene alone does not disrupt F-actin organization. When operating in cooperation with the SV40 large T antigen, however, it leads to the reorganization of F-actin at the cell periphery and confers a rounded structure on the cells.

Actins↗
PubMed1990

Preparation and crystallization of a human immunodeficiency virus p24-Fab complex.

A recombinant form of human immunodeficiency virus capsid protein, p24, expressed in Escherichia coli has been purified to homogeneity and separated into distinct isoelectric forms. A monoclonal antibody, mAb25.4, which recognizes an epitope in the amino-terminal region of p24, has been purified to homogeneity from ascites fluid and digested with papain to produce the respective antigen-binding fragment (Fab). The Fab25.4 was purified from the digestion mixture and separated into two distinct isoelectric forms. The two Fab species were each complexed with one isoelectric form of the recombinant p24 by incubating equimolar quantities of the two proteins. Two different crystal morphologies of the p24-Fab25.4 complex were obtained by the vapor-diffusion method with 12-24% PEG 3350 as the precipitant. One of these crystal forms has unit-cell parameters of a = 92.1 A, b = 85.4 A, c = 54.0 A, alpha = gamma = 90.0 degrees and beta = 90.4 degrees and belongs to the monoclinic space group P2(1), with one molecule of the complex per asymmetric unit. These crystals strongly diffracted x-rays to at least 2.7-A resolution.

Antibodies, Monoclonal↗
PubMed1990

Expression in Escherichia coli and purification of human immunodeficiency virus type 1 capsid protein (p24).

Capsid protein (p24;CA) of human immunodeficiency virus type 1 (HIV-1) was synthesized in Escherichia coli strain BL21 (DE3) using a plasmid encoding a truncated HIV-1 gag/pol gene. The plasmid, which contained a mutation in the frameshift region, expressed viral proteinase (PR), a pol gene product, in the gag reading frame, resulting in efficient processing of mature CA and other gag-related products. The expressed CA is soluble, recognized by monoclonal antibodies directed against HIV CA and has an N-terminal sequence identical to that of CA purified from HIV. Purification was done under mild conditions where coexpressed HIV PR retained enzymatic activity. Milligram quantities of 90% pure CA protein were obtained after chromatography on DEAE cellulose followed by facilitated aggregation of the CA in the unbound fraction. The precipitated CA was readily dissolved in low ionic strength aqueous buffer. Gel exclusion chromatography results indicated that, in solution, CA existed in oligomeric form.

Base Sequence↗
PubMed1990

Formation of N2,3-ethanoguanine in DNA after in vitro treatment with the therapeutic agent, N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea.

HPLC analyses of the bases released by acid from N-(2-chloroethyl)-N-nitrosourea-treated DNA and N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea-treated DNA show the presence of a new guanine adduct, N2,3-ethanoguanine. This derivative can be synthesized at the monomer level by treating 2-hydroxyethylguanine with thionyl chloride. The product of this reaction, purified by HPLC, has been shown to have a mol. wt corresponding to ethanoguanine by mass spectrometry; NMR spectrometry also supports this structural assignment. The UV and fluorescence spectra are very similar to those of N2,3-ethenoguanine, providing evidence that the ethano bridge is attached between N2 and 3 positions. Proof that the derivative is N2,3-ethanoguanine comes from the fact that it can be converted to N2,3-ethenoguanine by dehydrogenation on a palladium catalyst. The discovery of this new derivative raises to four the number of tricylic derivatives that have been isolated from DNA treated with 2-haloethylnitrosoureas. The new adduct, N2,3-ethanoguanine, is closely related to an etheno adduct formed by chloroacetaldehyde, a metabolite of the human carcinogen vinyl chloride, and may have relevance to either the therapeutic or carcinogenic actions of the 2-haloethylnitrosoureas.

Chromatography, High Pressure Liquid↗
PubMed1989

Activity of purified biosynthetic proteinase of human immunodeficiency virus on natural substrates and synthetic peptides.

Retroviral capsid proteins and replication enzymes are synthesized as polyproteins that are proteolytically processed to the mature products by a virus-encoded proteinase. We have purified the proteinase of human immunodeficiency virus (HIV), expressed in Escherichia coli, to approximately 90% purity. The purified enzyme at a concentration of approximately 20 nM gave rapid, efficient, and specific cleavage of an in vitro synthesized gag precursor protein. Purified HIV proteinase also induced specific cleavage of five decapeptide substrates whose amino acid sequences corresponded to cleavage sites in the HIV polyprotein but not of a peptide corresponding to a cleavage site in another retrovirus. Competition experiments with different peptides allowed a ranking of cleavage sites. Inhibition studies indicated that the HIV proteinase was inhibited by pepstatin A with an IC50 of 0.7 microM.

Amino Acid Sequence↗
PubMed1989

A comparison of the effects of the prostaglandin synthesis inhibitors indomethacin and carprofen on 7,12-dimethylbenz[a]anthracene-induced mammary tumorigenesis in rats fed different amounts of essential fatty acid.

The effects of the cyclooxygenase inhibitors indomethacin and carprofen on the enhancement of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis by dietary linoleate have been compared in female Sprague-Dawley rats. Indomethacin and carprofen, 0.004% and 0.02% (w/w) in the diet, respectively, were fed to rats receiving 20% fat diets containing 0.5, 4 or 12% linoleate starting 7 days after administration of 5 mg DMBA i.g. Indomethacin was shown to have a marked inhibitory effect on mammary tumorigenesis in rats fed the 4 and 12% linoleate diets, but did not alter tumorigenesis in rats fed the 0.5% linoleate diet. In contrast, carprofen was not inhibitory in any of these dietary groups, or in a separate experiment in which a 5% fat--3% linoleate diet was fed. The effect of each drug on prostaglandin E2 (PGE2) levels in normal mammary glands enriched in epithelial cells after a 3-week pretreatment with 17 beta-estradiol and progesterone was also investigated. Carprofen was shown to reduce PGE2 levels to a similar or greater extent than indomethacin at each level of linoleate in the diet. These data demonstrate that a reduction in PGE2 synthesis in the mammary epithelium does not correlate with inhibition of mammary tumorigenesis, and that other factors, including possible alterations in other products of the arachidonic acid cascade, are responsible for this inhibitory effect.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1989

The pharmacology of antiulcer drugs.

The use of medications for the treatment of gastrointestinal ulcers has evolved to a great extent since the early days of therapy with diet and antacids. Today a number of different agents are available to treat the causative factors of ulcer formation. Currently, antacids, histamine2-receptor antagonists, and sucralfate are considered frontline therapies suitable for most patients. The future also looks promising for newer agents, such as omeprazole and prostaglandin analogs. The purpose of this article is to provide practitioners with an understanding of the achieved more efficiently and effectively.

Anti-Ulcer Agents↗