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Biomedical subjects

B R Clark

Publications and source records attributed to B R Clark.

At least 55 records · Page 3Linked to original sources
PubMed1985

High-specific-activity 111In-labeled anticarcinoembryonic antigen monoclonal antibody: improved method for the synthesis of diethylenetriaminepentaacetic acid conjugates.

A new method has been developed for conjugating diethylenetriaminepentaacetic acid (DTPA) to proteins using the N-hydroxysuccinimide active ester of DTPA. The DTPA-active ester was prepared using diisopropylcarbodiimide in a simple single step synthesis. DTPA-conjugated proteins were prepared by adding the DTPA-active ester reaction mixture to protein solutions (5 mg/ml) buffered at pH 7.0 and purified by Sephadex G-50 chromatography. A monoclonal antibody directed against carcinoembryonic antigen was reacted with four different amounts of the DTPA-active ester. Solid-phase enzyme immunoassay showed that the immunological activity of the antibody conjugate was not altered when the active ester: antibody molar ratio was 36:1 or 72:1; however, it decreased when the ratio was 180:1 or 360:1. The antibody heavy and light chains had slightly decreased electrophoretic mobilities when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a result consistent with the covalent attachment of DTPA to the protein. Sephadex G-200 chromatography showed that the native and conjugated antibodies were the same size. When the DTPA-conjugated antibody was incubated with 10, 50, and 100 microCi of 111In/micrograms of protein, specific activities of 9.8, 43.1, and 56.3 microCi/micrograms were obtained. Enzyme immunoassay and radioimmunoassay of the 111In-labeled antibody showed that it retained its full immunological activity. The high specific activity of the 111In-labeled antibody makes it suitable for imaging carcinoembryonic antigen-bearing tumors using low doses of antibody.

Animals↗
PubMed1984

Use of biotin-labeled monoclonal antibodies and avidin-peroxidase conjugates for the determination of epitope specificities in a solid-phase competitive enzyme immunoassay.

The epitope specificities of monoclonal antibodies against carcinoembryonic antigen (CEA) were determined in a solid-phase, biotin-avidin enzyme immunoassay. Constant amounts of biotin-labeled antibodies were incubated with the immobilized antigen in the presence of decreasing amounts of unlabeled antibodies. The biotinylated antibodies bound to the antigen were then reacted with avidin-peroxidase conjugates. The activity of the bound peroxidase was determined by the use of o-phenylenediamine and hydrogen peroxide.

Animals↗
PubMed1984

Production of mitogen-free immune interferon and T-cell growth factor by human peripheral blood lymphocytes induced with biotin-labeled staphylococcal enterotoxin A.

A method for the facile removal of mitogens or inducers of lymphokine production from cell culture medium of stimulated cells is described. The technique is based on the covalent attachment of biotin to mitogen or inducer and the removal of the biotinylated products from stimulated cell culture medium using immobilized avidin. Using this procedure, biotin-labeled staphylococcal enterotoxin A (SEA-B) was shown not to differ significantly from unmodified SEA in its capacity to stimulate mitogenesis and induce production of immune interferon (IFN) and T-cell growth factor (TCGF) in cultures of human mononuclear cells from peripheral blood. SEA-B was also shown not to differ from SEA in its binding to SEA antibodies. Results of mitogenicity studies and competitive radioimmune assay (RIA) measurements indicate that SEA-B is essentially completely removed from stimulated cell culture medium by absorption with avidin coupled to Sepharose 4B.

Biotin↗
PubMed1984

Cloning and physical mapping of a gene fragment coding for a 64-kilodalton major late antigen of human cytomegalovirus.

We have isolated a clone containing a gene fragment coding for a 64-kilodalton glycoprotein that is the major late antigen of human cytomegalovirus (HCMV). Based upon the amino acid sequence of a tryptic peptide of this glycoprotein (HCMVgp64), two sets of mixed-sequence probes, one consisting of a mixture of 16 heptadecadeoxyribonucleotides and the other a mixture of 32 icosadeoxyribonucleotides, were synthesized. A subgenomic library of HCMV (Towne strain) DNA was constructed in plasmid pBR327 and transformants were screened with 32P-labeled aliquots of these synthetic oligodeoxyribonucleotide probes. Two clones among 15,000 gave strong positive signals. Plasmid DNA was isolated from the positive clones and characterized by restriction mapping and Southern blot analysis using both probes. The plasmid DNA contained a 2.3-kilobase insert, which yielded an 800-base-pair and a 1500-base-pair fragment after Sau3A digestion. Only the 800-base-pair fragment hybridized to the mixed probes, and DNA sequence analysis revealed that it contains nucleotide sequences compatible with amino acid sequences of tryptic peptides of HCMVgp64. Restriction mapping studies of HCMV DNA using this 32P-labeled 800-base-pair cloned DNA have allowed us to locate this gene fragment in the long unique region of HCMV (Towne strain) genome at approximately equal to 0.5-0.51 map unit.

Antigens, Viral↗
PubMed1984

Isolation and partial chemical characterization of a 64,000-dalton glycoprotein of human cytomegalovirus.

A guanidinium chloride extract of [3H]glucosamine- and [35S]methionine-labeled virions plus dense bodies of human cytomegalovirus (Towne) was separated by reverse-phase high-pressure liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the eluate revealed the major peak to be a glycoprotein with a relative mass of 64,000. This glycoprotein (HCMVgp64) was characterized by amino acid analysis and a high-pressure liquid chromatographic map of its tryptic peptides.

Amino Acids↗
PubMed1983

Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: determination of affinities and specificities of monoclonal antibodies by using biotin-labeled antibodies and avidin as precipitating agent in a solution phase immunoassay.

A general method is described for the determination of affinity constants and antigen cross-reactivities of monoclonal antibodies. The method employs biotin-labeled antibody, radiolabeled antigen, and avidin as a precipitating agent in a homogeneous phase, competitive radioimmunoassay. This method eliminates incomplete or variable precipitation of antigen-antibody complexes often encountered in immunoassays in which monoclonal antibodies are employed. Using this assay system, we were able to rapidly determine the affinity constants for a number of monoclonal antibodies elicited to carcinoembryonic antigen (CEA). In the preceding paper it was shown that five of the monoclonal antibodies recognized distinct epitopes on CEA. In antigen-binding experiments with these five monoclonal antibodies, the percent of radiolabeled CEA bound in antibody excess ranged from 30 to 92%. The CEA cross-reacting antigens, normal cross-reacting antigen (NCA), and tumor-extracted, CEA-related antigen (TEX) were significantly bound by one, and to a lesser degree, by two of the five antibodies. Two antibodies did not bind significant amounts of NCA or TEX. In inhibition studies, the amount of unlabeled CEA leading to 50% inhibition of 125I-labeled CEA-binding was in the range of 3.7 to 760 ng per tube. The amount of TEX showing the same degree of inhibition was 23-fold greater than the amount of CEA for two antibodies and 351-fold greater than the amount of CEA for a third antibody. The affinity constants for CEA were in the range of 1.0 x 10(8) to 5.1 x 10(10) M-1. The affinity constants for NCA and TEX, determined for one of the antibodies, were three orders of magnitude lower in comparison to CEA. The heterogeneity of radiolabeled CEA as indicated by the low fraction bound by one of the monoclonal antibodies is shown to be most probably an artifact resulting from radioiodination damage. The application of the approach described in this report should eliminate the problems most commonly encountered in the determination of affinity constants for monoclonal antibodies or the use of monoclonal antibodies in competitive, homogeneous-phase immunoassays.

Animals↗
PubMed1982

Embryotoxic and teratogenic effects of aqueous extracts of tar from a coal gasification electrostatic precipitator.

Aqueous extracts of tar from a coal gasification electrostatic precipitator were tested for its toxic and teratogenic potential in vitro on embryos of the amphibian Xenopus laevis. The 96-h LC50 and EC50 were determined to be 0.83% and 0.48%, respectively. The developmental stage of normal-appearing exposed embryos is not affected by increasing concentrations of the extract. Embryo growth, however, is significantly reduced at concentrations as low as 0.25%. Motility and pigmentation were effectively reduced relative to controls by extract concentrations of 0.5% and greater. Exposed embryos are shorter and stockier than controls. Malformations of head, eyes, viscera, and spine are common, and cartilage formation is abnormal. The epidermis is often hyperplastic, and large blisters occur over the somatic surface. The severity of abnormal development is directly related to the concentration of the toxicant to which the embryos are exposed. Chemical analysis shows that the aqueous extracts contain phenols, furans, monoaromatic and diaromatic hydrocarbons, and mono- and diazaarenes and/or monoaromatic amines.

Abnormalities, Drug-Induced↗
PubMed1982

Chemical synthesis of urotensin II, a somatostatin like peptide in the caudal neurosecretory system of fishes.

In the goby, Gillichthys mirabilis, urotensin II (a bioactive neuropeptide present in the urophysis of teleost fish) has the dodecapeptide sequence, H2N-AGTADC-FWKYCV-OH, which is homologous with mammalian somatostatin at positions 1, 2 and 7-9. The Merrifield solid phase synthesis of Gillichthys urotensin II (UII) was accomplished by stepwise assembly from the carboxy terminus using N-alpha-tert, butyloxycarbonyl (Boc) amino acids containing benzyl-derived groups for protection of side-chain functionalities, Coupling of amino acids to the growing peptide was mediated by diisopropylcarbodiimide (DIC) in the presence of 1-hydroxybenzotriazole (HOBt). Residual alpha-amino groups remaining after coupling were blocked by acetylation with 1-acetylimidazole. Crude, synthetic UII was extracted from the HF-treated, protected peptide-resin product, reduced with dithiothreitol (DTT), reoxidized at high dilution with O2, and separated into its components using a single, preparative, reverse-phase HPLC step. The pure, synthetic UII, obtained in 7.6% yield from oxidized crude UII, was indistinguishable from pure, native UII in specific bioactivity, amino acid sequence, and retention time in each of two different HPLC systems.

Amino Acid Sequence↗
PubMed1981

Analytical and biological analysis of test materials from the synthetic fuel technologies.

Nitrogen-containing organic compounds from environmental sources are receiving increasing attention because of uniquely active mutagens which have been found in this class (Chrisp et al., 1978; Nagao and Sugimura, 1978: Guerin et al., 1980) Differences in mutagenic activities among the various organo-nitrogen compounds, i.e., pyrrole types, pyridine types and aniline types, have been noted consistently. Furthermore, differences among homologs of a particular compound type are often striking. Information in this paper engages the question of chemical structure/biological activity relationships. Activity data for several N-heterocyclic, nitro-, amino- (primary, secondary and tertiary), and amino-N-heterocyclic aromatic compounds are presented. The number of fused rings and the substituent type affect the mutagenic activities greatly. The trends observed are discussed generally with reference to molecular structural features.

Cyclic N-Oxides↗
PubMed1981

Analytical and biological analyses of test materials from the synthetic fuel technologies. III. Use of sephadex LH-20 gel chromatography technique for the bioassay of crude synthetic fuels.

To determine the health effects associated with newly emerging energy technologies, we have subjected a group of synthetic fuels to mutagenicity evaluation, using the Ames Salmonella assay. Coupling of chemical fractionation to the mutagenicity assays was necessary. Fractions obtained by use of Sephadex LH-20 gel chromatography on crude-coal-derived oils and shale oil were tested for mutagenicity with strain TA98 (with Aroclor S9 mix). Mutagenicity results obtained with synthetic fuels were compared with those from a mixture of natural petroleum crude oils. Merits of the Sephadex LH-20 separation technique and precautions in interpreting experimental results are discussed.

Chromatography, Gel↗
PubMed1981

Relative potency estimation for synthetic petroleum skin carcinogens.

A procedure for quantitative analysis of skin carcinogenesis data, for the purpose of establishing carcinogenic potency, has been applied to observations obtained from C3H mice exposed continuously to synthetic and natural petroleums. The importance of total polynuclear aromatic (PNA) content to the skin carcinogenic activity of the crude materials was also examined. Of three synthetic petroleums evaluated, all were shown capable of inducing skin neoplasms within a two-year exposure period. Under comparable exposure conditions a composite sample of five natural petroleums was noncarcinogenic. Comparison of the distributions of times to initial skin neoplasm versus dose rate, for groups exposed to synthetic fossil liquids and the reference skin carcinogen, benzo(a)pyrene, provided estimates of relative carcinotenic potency for the synthetic petroleums ranging from 1/500 to 1/1400 the potency of benzo(a)pyrene. The carcinogenic activity of a chemically isolated PNA fraction versus the crude from which it was derived suggested that this fraction was responsible for the carcinogenic activity of these synthetic petroleums in mouse skin.

Animals↗
PubMed1981

Sexual maturation and fecundity of wild and domestic Norway rats (Rattus norvegicus).

A population of captive-reared wild Norway rats and two stocks of domestic rats were compared for various parameters of sexual maturation and fecundity. Males did not differ in age at the first appearance of spermatozoa in the testes but wild males were significantly older than Long--Evans and Sprague--Dawley domestic males at first copulation with a hormone-induced oestrous female. Wild females were significantly older than domestic females at the time of vaginal opening, first oestrus and first conception. Stocks did not differ in length of the first oestrous cycle. Wild females produced significantly smaller litters than their domestic counterparts. Domestic rats were significantly heavier than wild rats at the various stages of sexual maturation. The results support the hypothesis that domestication of the rat has been accompanied by genetic and/or environmental changes that accelerate sexual development and improve breeding success under laboratory conditions.

Age Factors↗
PubMed1980

Urotensin II: a somatostatin-like peptide in the caudal neurosecretory system of fishes.

Urotensin II, a peptide hormone from the caudal neurosecretory system of the teleost, Gillichthys mirabilis, was isolated by using classical chromatographic techniques and high-performance liquid chromatography (HPLC). Direct microtechniques for sequence determination were used to establish its structure. Urotensin II from Gillichthys is a 1363-dalton dodecapeptide with the amino acid sequence Ala-Gly-Thr-Ala-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val. This sequence is homologous with somatostatin in positions 1 and 2 and 7-9. The sequence has been verified by the production of a bioactive synthetic urotensin II. The possible chemical and physiological significance of its homology to somatostatin is discussed.

Amino Acid Sequence↗