Biomedical subjects

D C Graves

Publications and source records attributed to D C Graves.

13 recordsLinked to original sources

PubMed1992

Evidence for secretion of high molecular weight mucins by canine tracheal epithelial cells in primary culture: effects of select secretagogues in mucin secretion.

The purpose of this investigation was to provide evidence for the secretion of high molecular weight mucins, CTM-A and CTM-B, in primary culture of canine tracheal epithelial (CTE) cells. The cells were isolated from tracheas of mongrel dogs by pronase treatment. Primary cultures of the epithelial cells were established using ICN collagen inserts in Dulbecco's modified Eagle's/F12 medium supplemented with growth factors and could be maintained for up to 23 days. The evidence for the mucin secretion in culture medium and their localization in the cells was established by a) positive immunocytochemical staining using specific antibodies developed against purified native as well as deglycosylated CTM-A and CTM-B; b) incorporation of labeled amino acids, followed by electrophoresis and autoradiography detection of glycoconjugates purified from the culture medium; c) comparison of the amino acid compositions of mucin purified from canine tracheal pouch secretions and that purified from the culture medium; and d) Western blot analyses using specific polyclonal antibodies directed against deglycosylated CTM-A and CTM-B. Immunoaffinity purified secreted labeled glycoconjugates were resistant to hyaluronidase treatment. The effects of cyclic AMP (1 x 10(-5) M), dibutyryl cyclic AMP (1 x 10(-5) M), 8-bromocyclic AMP (1 x 10(-5) M), and prostaglandin E1 (1 x 10(-6) M) on mucin secretion by CTE cells were also investigated. Secretion of mucins by CTE cells in culture was considerably more enhanced by 8-bromocyclic AMP than that observed for other secretagogues used in this study.

Alprostadil↗

PubMed1992

Cell-growth quantitation methods for the evaluation of antiestrogens in human breast cancer cells in culture.

The antiproliferative activity of the antiestrogen, tamoxifen, on the growth of MCF-7 human breast cancer cells was evaluated using the hemocytometric trypan blue exclusion method, [3H]-thymidine incorporation, and a total protein determination. Tamoxifen was evaluated over a concentration range from 10(-9) to 10(-6) M. The hemocytometric trypan blue exclusion method and [3H]-thymidine incorporation were sensitive enough to demonstrate the inhibitory influence of tamoxifen on the proliferation of MCF-7 cells at a concentration as low as 10(-9) M. A very good correlation of these two methods was observed in the submicromolar concentration range of tamoxifen. The total protein determination method was only sensitive enough to detect the antiproliferative influence of tamoxifen at concentrations above 10(-6) M. In conclusion, the [3H]-thymidine incorporation method was found to be effective and much less time consuming than the hemocytometric trypan blue exclusion method for evaluating the antiproliferative effects of antiestrogens in cultured MCF-7 cells. However, when evaluating antiestrogens, which are cell-cycle specific, the results of the [3H]-thymidine incorporation method should be interpreted with caution.

Algorithms↗

PubMed1992

Comparative study of antipneumocystis agents in rats by using a Pneumocystis carinii-specific DNA probe to quantitate infection.

A repetitive genomic DNA clone (B12-2) that specifically hybridizes to Pneumocystis carinii DNA has been identified. No cross-hybridization to genomic DNA prepared from bacteria, other fungi, protozoa, or mammals was observed. Clone B12-2 is multiply represented in the P. carinii genome. By direct hybridization to DNA prepared from the lungs of immunosuppressed rats, the probe can detect the equivalent of fewer than 1,000 P. carinii organisms. A hybridization assay employing clone B12-2 has been developed to quantitate organism load in the rat model for P. carinii. Application of the assay to track the accumulation of organisms during the immunosuppression regimen as well as to monitor the efficacy of two drug therapies used clinically for the treatment of P. carinii pneumonia is described here. The clone B12-2 hybridization assay for the determination of P. carinii organism load possesses several advantageous features and thus should serve to complement conventional staining and immunohistochemical methods.

Animals↗

PubMed1992

Synthesis and biological evaluation of a series of gem-dichlorocyclopropanes as antitumor agents.

As part of our continuous effort to produce non-steroidal antiestrogens demonstrating less intrinsic estrogenicity and greater antagonism than those in use, a series of Analog II (1,1-dichloro-2,3-diphenylcyclopropanes) derivatives was synthesized. The compounds were tested for their ability to inhibit the growth-stimulating action of estradiol in the immature mouse uterus and estrogen receptor (ER) (+) MCF-7 human breast cancer cells in vitro. Like Analog II, the derivatives were found to have no intrinsic estrogenicity (except 30) and they antagonized estradiol action less completely than the lead compound. Polarity improved the ER binding affinity of Analog II, but was quite small for all compounds, except 30, for which it was comparable to tamoxifen. Six compounds (8, 10, 14, 23, 29 and 30) demonstrated antiproliferative activity toward MCF-7 cells, in vitro, and the mean inhibition period over 6 days ranged from 20 to 37%. Only compound 30 was reversed by estradiol.

Animals↗

PubMed1991

Detection of distinct species in purified human respiratory mucin using monoclonal antibodies.

The purpose of this investigation was to demonstrate the presence of different species (subpopulations) in the purified human tracheobronchial mucin (HTM-1). Mucin was highly purified from sputum specimens collected from a cystic fibrosis (CF) patient using a protocol involving sequential chromatography on Bio-Gel A-5m and hydroxylapatite columns. SDS-composite gel electrophoresis followed by periodic acid-Schiff's reagent staining was unable to detect mucin species. However, using enzyme-linked immunoelectrotransfer blot (EITB) method and polyclonal antibodies raised against HTM-1, at least four different migrating mucin species were detected. Further immunological characterization of these mucin species was carried out using a library of 16 monoclonal antibodies (MAbs) developed against the purified mucin. Nine MAbs belonged to the IgM class, two MAbs were IgG1, one IgG2a and remaining four were of the IgG3 subclass. Periodate oxidation of the mucin antigen was used to establish the nature of the mucin epitopes recognized by the MAbs. 11 MAbs recognized carbohydrate epitopes in the mucin molecule that were sensitive to periodate, while five MAbs reacted with periodate resistant carbohydrate epitopes or the protein portion of the mucin molecule. Enzyme-linked immunoelectrotransfer blot analysis of the MAbs against HTM-1 showed the presence of at least three distinct mucin species. Chromatography of the mucin on immunoaffinity columns (MAbs H(13.3), M(33.3) and CCK 061 conjugated to CNBr-activated Sepharose 4B), followed by ELISA and EITB analyses, established the mucin species recognized by the antibodies. These experiments further indicated that both unique and shared epitopes were present in the mucin species. These monoclonal antibodies may provide a promising approach to differentiate the secretory products of the tracheobronchial tree.

Antibodies, Monoclonal↗

PubMed1991

Production and characterization of monoclonal antibodies to purified deglycosylated cystic fibrosis respiratory mucin: evidence for the presence of four immunologically distinct epitopes.

Respiratory mucus glycoproteins (mucins) were purified from the tracheobronchial secretions of three Cystic Fibrosis (CF) patients. The mucins were completely deglycosylated by treatment with trifluoromethanesulfonic acid and subsequent treatment with alpha-N-acetylgalactosaminidase. Over thirty hybrid clones secreting antibodies against the deglycosylated mucin (DGM) were obtained using standard hybridoma techniques. Hybrids with positive identification for CF-DGM were cloned twice using limiting dilution method to ensure the monoclonal nature of the antibodies. Eight stable clones (1a, 1b, 10a, 10c, 10d, 10e, 29d, and 30e) secreting monoclonal antibodies (MAbs) showing specificity of reaction to CF-DGM were obtained. Two clones, 29d and 30e, secreted antibodies of the IgM class while the other six clones secreted antibodies of the IgG1 subclass. Denaturation and reduction experiments suggested that MAbs 1b, 10e, 29d and 30e were directed against a given sequence of amino acids in the DGM while the other four MAbs, in addition to being sequence specific, were also conformation dependent. Further, competitive binding radioimmunoassays suggested that MAbs 1b, 10e, 29d and 30e recognize four distinct epitopes in the peptidic core of CF respiratory mucin. In summary, the MAbs may provide a promising approach to elucidate the structure of the polypeptide backbone of human respiratory mucins as well as for the screening of cDNA libraries for clones secreting mucin(s).

Amino Acids↗

PubMed1991

Antiproliferative activity of a series of novel cyclopropyl antiestrogens on MCF-7 human breast cancer cells in culture.

The potential antitumor activity of a series of novel cyclopropyl compounds, which lack estrogen agonist activity, was evaluated in estrogen receptor positive human breast cancer cells (MCF-7) in culture. The compounds were evaluated to determine their antiproliferative activity at a concentration of 1 microM at 2, 4 and 6 days of treatment by hemocytometer using the Trypan Blue exclusion method to count viable cells. Estradiol-induced reversibility of the antiproliferative activity of these compounds was also evaluated. The activity of a series of 19 diaryl- and triarylcyclopropyl compounds was examined. Thirteen compounds inhibited the growth of MCF-7 cells while six were inactive. Five of the 13 active compounds produced antiproliferative activity which was reversed by 0.1 microM estradiol. Thus, several of these novel cyclopropyl compounds may be useful in the treatment of hormone-dependent breast cancer and other estrogen-dependent tumors.

Antineoplastic Agents↗

PubMed1977

Inhibition of the reverse transcriptase of bovine leukemia virus by antibody in sera from leukemic cattle and immunological characterization of the enzyme.

Sera from some leukemic cattle contain an antibody that inhibits the reverse transcriptase activity of the bovine leukemia virus. The antibody is not directed against the synthetic template or the major internal and envelope viral antigens. The antibody failed to inhibit the DNA polymerases of the murine leukemia virus, simian sarcoma-associated virus, avian myeloblastosis virus, or Escherichia coli. Conversely, the bovine leukemia virus enzyme was not inhibited by antibody against the reverse transcriptases of other C-type viruses. These findings agree with previous results showing that the major internal bovine leukemia virus protein lacks the known interspecies- and intraspecies-specific antigenic determinants indentified in the homologous proteins of other oncornaviruses.

Animals↗

PubMed1977

A reverse transcriptase assay for detection of the bovine leukemia virus.

An RNA-dependent DNA polymerase or reverse transcriptase has been demonstrated in highly purified bovine leukemia virus (BLV) particles. The viral enzyme responded very effectively to the exogenous template primer polyneucleotide (poly) (rA)-oligonucleotide (oligo) (dT). Unlike the reverse transcriptases of most mammalian C type RNA viruses, and of the ubliquitous foamy-like bovine syncytial virus, the BLV enzyme prefers magnesium rather than manganese for optimal activity. The identification of several other conditions required for optimal activity of the viral reverse transcriptase led to the development of a rapid, sensitive, semiquantitative assay, which is comparable in sensitivity to the syncytia-infectivity assay for the detection of BLV in supernatant fluids of monolayer cell cultures. However, the reverse transcriptase assay is not sufficiently reproducible for obtaining routine detection of BLV in short-term cultures of bovine peripheral blood lymphocytes. Therefore, this assay does not seem to provide an accurate method for the diagnosis of BL virus infection in cattle.

Cells, Cultured↗

PubMed1976

Bovine leukemia virus genes in the DNA of leukemic cattle.

Reverse transcripts of the rna genome of the bovine leukemia virus (BLV) as well as 125I-labeled BLV RNA hybridize to the DNA of tissues from leukemic cattle with the adult form of the disease but not to bovine thymic lymphoma or normal bovine tissues.

Animals↗

PubMed1976

Isolation and characterization of an antigen of the bovine C-type virus.

By means of gel filtration and isoelectric focusing, an antigen of the bovine C-type leukemia virus was isolated in a highly purified form from extracts of infected cells. The antigen has a molecular weight of approximately 25,000 daltons and an isoelectric point of 6.4 to 6.6. In immunodiffusion experiments, the antigen forms a line of identity with an antigen extracted from highly purified bovine C-type leukemia virus by treatment with ether or Triton X-100. As determined by immunodiffusion analyses, the bovine C-type leukemia virus antigen does not have antigenic determinants in common with the murine or feline leukemia viruses, the foamy-like bovine syncytia virus, or the Mason Pfizer monkey virus.

Antigens, Viral↗

PubMed1976

In vitro transmission and propagation of the bovine leukemia virus in monolayer cell cultures.

This study demonstrates that the bovine leukemia virus (BLV) can infect in vitro cells of human, simian, bovine, canine, caprine, ovine, and bat origin. Cultures of these cells, cocultivated with BLV-infected cells or inoculated with cell-free BLV preparations, continuoously showed the presence of cells with the internal BLV antigen as well as BLV-induced syncytia. Virus replication was abundant and increased with passage in bat lung cells and was moderate but constant in fetal canine thymus cells. The amounts of virus released by the simian DBS-FRhL-1 and caprine S-743 cultures were low to moderate during the first 4 to 8 weeks and decreased thereafter. In the infected fetal lamb spleen cell cultures, virus production was low and declined further with passage. Bovine embryonic spleen and human diploid embryonic lung WI-38 cell cultures produced very small amounts of virus only during the first two passages after inoculation despite the fact that they remained infected, as determined by the continuous presence of cell BLV antigen and syncytia. Morphologically and antigenically, the virus particles released by the monolayer cell cultures were indistinguishable from those found in short-term and long-term cultures of BLV-infected bovine lymphoid cells. Repeated electron microscopic examinations and serological tests showed that all the BLV-infected cultures, including those from which the infecting inocula were obtained, were free of the foamy-like bovine syncytial virus, parainfluenza 3 virus, infectious bovine rhinotracheitis virus, bovine viral diarrhea virus, and the maedi-like bovine R-29 virus.

Animals↗

PubMedDate not supplied

Ultrastructural localization of epitopes recognized by monoclonal antibodies produced to rat Pneumocystis carinii.

The binding sites of five monoclonal antibodies (MAb) developed against rat Pneumocystis carinii were examined at the ultrastructural level by using a post-embedding labeling method. Although all five MAb reacted with the pellicle of P. carinii, they were divided into two groups by localization of binding sites. The MAb 168.2.1, 174.2.1, and 215.2.1 reacted mainly with the electron-dense outer layer, whereas MAb 227.1.1 and 228.1.1 labeled both the outer dense layer and the middle lucent layer. With in the first group of MAb, no significant differences were observed in the reactivity patterns seen with the different stages of P. carinii. In the second group, however, the intensity of labeling of the electron-dense layer was higher in the precyst, cyst, and ruptured cyst stages than in the trophozoite stage. These latter results indicate that there may be an increase in antigen accumulation during development from the trophozoite to the cyst stages, or that antigens may be modified the development.

Animals↗