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R J Bjercke

Publications and source records attributed to R J Bjercke.

32 records · Page 2Linked to original sources
PubMed1987

Comparison of monoclonal and polyclonal antibodies to cotinine in nonisotopic and isotopic immunoassays.

Monoclonal antibodies (McAb) were used to develop nonisotopic and radioimmunoassays (RIA) for quantitative determination of the major nicotine metabolite, cotinine, in physiological fluids. ELISAs and fluorescence immunoassays were carried out in microtiter plate wells coated with a conjugate of cotinine 4'-carboxylic acid bound covalently to poly-L-lysine. The detection systems were horseradish peroxidase (HRP)-labeled staphylococcal protein A, HRP-streptavidin-biotin, and biotinylated alkaline phosphatase-4-methylumbelliferyl phosphate. With the three McAb tested, I50 values ranged between 0.024-0.063 ng cotinine and as little as 0.005-0.015 ng gave 15% inhibition. These assays were 5-20 times more sensitive than similar assays using six rabbit antisera. With McAb the standard inhibition curves were steeper and complete inhibition of immune binding was achieved with approximately 1 ng cotinine. In contrast, 100-500 ng cotinine failed to give greater than 80-90% inhibition with rabbit antibodies either in the plate assays or in RIA using a 125I-labeled tyramine derivative of cotinine as the tracer. In this RIA, the sensitivity with McAb (mean I50 of 0.55 ng cotinine) was over three-fold greater than with rabbit antisera (mean I50 of 1.84 ng). The presence of antibodies directed to the amide linkage group common to the polylysine conjugate. 125I-tyramine derivative and the immunogen likely accounts for the inferior quality of assays using rabbit antisera. Consistent with this conclusion, superimposable inhibition curves were obtained in the RIA when monoclonal or rabbit antibodies were used with [3H]cotinine. Cotinine levels in saliva, serum and plasma from smokers and non-smokers determined with McAb-based assays showed a strong correlation with values obtained by RIA using rabbit antisera or by gas chromatography. Properly selected McAb offer distinct advantages over conventional antisera in nonisotopic immunoassays and RIAs for cotinine as a biochemical marker of active or passive smoking.

Antibodies↗
PubMed1986

Stereospecific monoclonal antibodies to nicotine and cotinine and their use in enzyme-linked immunosorbent assays.

Stereospecific monoclonal antibodies (McAb) have been prepared against the tobacco alkaloid (S)-(-)-nicotine and its major metabolite (S)-(-)-cotinine. Nine anti-nicotine and 4 anti-cotinine hybridomas, selected by a screening procedure that utilized immunoprecipitation of the 3H-labeled natural isomers of nicotine or continine, were grown in the ascites fluid of pristane-primed syngeneic BALB/c mice. Antibodies in concentrations up to 7.5 mg/ml ascites and with binding affinities that generally exceeded 10(8) M-1 were obtained. Enzyme-linked immunosorbent assays (ELISAs) were developed in which nicotine or cotinine derivatives bound covalently to poly-L-lysine were coated onto wells of polyvinyl chloride microtiter plates. Coated wells were incubated sequentially with McAb in the presence or absence of inhibitor, rabbit anti-mouse immunoglobulin, then horseradish peroxidase-labeled protein A (HRP-SpA) before addition of substrate. The antibodies are highly specific and show minimal cross-reactivity with several nicotine metabolites and other structurally related compounds. In the respective assays, only 0.25 ng (S)-(-)-nicotine and 0.12 ng (S)-(-)-cotinine are required to give 50% inhibition of antibody binding, and as little as 0.05 ng nicotine and 0.02 ng cotinine give 15% inhibition. These assays are 5-10 times more sensitive than analogous ELISAs developed with rabbit antisera and HRP-SpA or conventional radioimmunoassays (RIAs) that utilize the rabbit antisera and 3H-labeled ligands. There was good correlation between the levels of nicotine (r = 0.967) and cotinine (r = 0.981) found in saliva samples from smokers and non-smokers assayed by McAb-based ELISAs and conventional RIAs.

Antibodies, Monoclonal↗
PubMed1984

Estrogen-induced 24K protein in MCF-7 breast cancer cells is localized in granules.

We have previously reported the production of monoclonal antibodies which detect, by immunohistochemistry, an estrogen-induced protein of molecular weight 24,000 daltons (24K). This protein, of unknown function, has been detected in: a) estrogen receptor-positive breast cancer cell lines but not in receptor-negative lines; b) several human normal estrogen target organs; and c) certain human carcinomas, including breast tumors. To examine the subcellular localization of this 24K estrogen-induced protein, we have done immunohistochemical studies at light and electron microscopic levels using a human breast tumor cell line (MCF-7) grown in vitro and also in nude mice in vivo. MCF-7 cells grown in the ascites fluid of nude mice and processed for paraffin sections showed a defined polarity, and the 24K protein was localized in the apical cytoplasm of the cells. After cytocentrifugation, MCF-7 cells grown in vitro displayed 24K protein mainly confined to large cytoplasmic granules. The presence of 24K protein in cytoplasmic granules was also seen by immunoelectronmicroscopy in MCF-7 cells grown both in vitro and in vivo. The granules had different sizes, shapes, and 24K immunostaining intensity. The morphological evidence suggests that the 24K estrogen-induced protein is secreted from the cells.

Animals↗
PubMed1984

Development of methods to measure activity of polysomes and cytoplasmic enzymes from bovine skeletal muscle in in vitro protein-synthesis assays.

A cell-free, protein-synthesis system containing components from bovine skeletal muscle was developed as prerequisite to attempts to learn whether polysomes or cytoplasmic enzymes limit rate of muscle protein synthesis in the current population of domestic animals. Amino acid incorporation into trichloroacetic acid-precipitable protein was optimal at pH 7.5 and, if Tris buffer was used, at K+ and Mg2+ concentrations of 40 mM and 4 mM, respectively. Optimal concentrations of compounds that provide energy for amino acid incorporation were 1 mM ATP, .2 mM GTP, 20 micrograms creatine phosphokinase/ml and 20 mM creatine phosphate; .03 mM of each of the 20 amino acids was required for assays lasting up to 60 min. Neither rate of tRNA acylation nor availability of aminoacyl-tRNA was rate-limiting in the cell-free system established; hence, the cytoplasmic enzyme fraction from bovine skeletal muscle contains ample tRNA and has aminoacyl-tRNA synthases that are sufficiently active to form aminoacyl-tRNA faster than these compounds are used to form polypeptides in the cell-free system developed. Reinitiation of ribosomes onto new mRNA occurred very slowly, if at all, in the protein-synthesis system developed. Cytoplasmic enzymes were rate-limiting whenever cytoplasmic enzyme protein to polysomal protein ratios were 3.2:1 or lower in the cell-free system. Polysomes were rate-limiting whenever cytoplasmic enzyme protein to polysomal protein ratios were 400:1 or higher in the cell-free system. These ratios define the conditions needed to assay activity of cytoplasmic enzymes or polysomes from different animals quantitatively.

Adenosine Triphosphate↗
PubMed1984

Relative roles of polysomes and cytoplasmic enzymes in regulating bovine skeletal muscle protein synthesis.

Activities of cytoplasmic enzyme or polysomal fractions, both prepared from longissimus muscle of bovine animals differing markedly in muscle growth were measured in cell-free, protein synthesis assays. Two groups of animals were used: 1) eight animals comprising two "double-muscled" animals fed a high-energy diet, two "double-muscled" animals fed a low-energy diet, two "normal" animals fed a high-energy diet and two "normal" animals fed a low-energy diet and 2) six animals differing in age and sex and including two bull calves, 87 and 145 kg; two 18- to 24-mo-old animals, 411 and 596 kg and two cows, 594 and 681 kg. Ability of cell-free, protein-synthesis systems prepared from muscle of different animals to incorporate amino acids into TCA-precipitable protein differed significantly and was in direct correlation with phenotypic muscle growth. Mixing experiments using cytoplasmic enzymes from one set of animals with polysomes from another set showed that source of cytoplasmic enzymes had a greater effect on rate of in vitro protein synthesis than source of polysomes. Cytoplasmic enzymes and polysomes from double-muscled animals and animals fed a high-energy diet supported more rapid in vitro protein synthesis than the same components from normal animals or animals fed low-energy diets. The "double-muscling" characteristic had a greater effect on rate of in vitro protein synthesis than did high or low-energy diets. Cytoplasmic enzymes and polysomes from calves supported a greater rate of in vitro protein synthesis than the same components from 18- to 24-mo-old animals or cows. Based on these limited results in one species and with two groups of animals in that species, cytoplasmic enzymes may have a greater role in limiting rate of muscle protein synthesis in the current population of domestic animals than polysomes. Composition of the crude cytoplasmic enzyme fraction used in this study is unknown, but it likely contains elongation and termination factors in addition to aminoacyl-tRNA synthases and regulatory RNA.

Age Factors↗
PubMed1983

Monoclonal antibody storage conditions, and concentration effects on immunohistochemical specificity.

Monoclonal antibodies against a 24,000 dalton intracellular estrogen-regulated protein in human breast cancer cells were used to study storage conditions and the effects of monoclonal antibody concentrations on immunohistochemical antigen localization. Both hybridoma supernatants and ascites fluid obtained from mice injected with hybridoma cells were used as sources of monoclonal antibodies; the monoclonal antibodies in the ascites fluid were concentrated and purified. Both antibody preparations were stored at 4, -20, or -70 degrees C and periodically tested for activity at these storage conditions. There was no difference in activity for the antibodies between storage at -20 and -70 degrees C. However, when highly diluted antibody was stored at 4 degrees C, the activity was lost within 2 weeks if carrier proteins were not added. These monoclonal antibodies were applied to immunohistochemical staining of different mouse and human tissues processed for routine paraffin sections, using the avidin-biotin-peroxidase procedure. A monoclonal antibody of unrelated specificity was used as control. When these antibodies were used at high concentrations, all the different tissues examined were immunostained. With reduction of the antibody concentration, an immunohistochemical dissection of the tissues was seen until specific immunostaining was reached. When even more highly diluted monoclonal antibody was used, heterogeneity in the staining pattern became very high. On the basis of these results, certain immunohistochemical criteria are proposed for the selection of the optimum concentration of monoclonal antibodies for specific antigen detection.

Animals↗
PubMed1983

Detection of a Mr 24,000 estrogen-regulated protein in human breast cancer by monoclonal antibodies.

Monoclonal antibodies have been prepared that can specifically detect an estrogen-regulated protein with a molecular weight of 24,000 in the MCF-7 human breast tumor cell line and in human breast tumor biopsies. These antibodies are of the immunoglobulin G1 subclass, exhibit a high affinity for the Mr 24,000 protein, and recognize an antigenic site that is stable to sodium dodecyl sulfate gel electrophoresis and electrophoretic transfer techniques. These monoclonal antibodies have been used to confirm estrogen regulation of the Mr 24,000 protein in MCF-7 cells and to detect Mr 24,000 protein in certain human breast tumors.

Animals↗
PubMed1982

Immunohistochemical detection of an estrogen-regulated protein by monoclonal antibodies.

It has been shown previously that estradiol regulates the synthesis of a Mr 24,000 cytosol protein in MCF-7 human breast cancer cells. This protein is an attractive probe to study the mechanisms of estrogen regulation in human breast cancer. We have produced monoclonal antibodies against the Mr 24,000 protein which show immunocytochemical localization in the cytoplasm of MCF-7 cells grown as solid tumors in nude mice. They do not react with normal mouse tissue or with MDA-MB-231 cells, an estrogen receptor-negative cell line. Moreover, the monoclonal antibodies react against certain human breast tumor biopsy samples. These data suggest that the monoclonal antibodies obtained will be useful to detect the estrogen-regulated protein by immunohistochemistry.

Antibodies, Monoclonal↗
PubMed1981

Codon binding and translational properties of an isoaccepting lysine tRNA peculiar to virus-transformed Cells.

Isoaccepting lysyl-tRNAs from virus-transformed cells in culture were fractionated in the RPC-5 system into peaks 1, 2, 4, 5a, 5, and 6. tRNALys6 previously was found predominantly associated with transformed cells. The codon response of each peak was determined in an E. coli ribosomal binding assay. tRNALys1, tRNALys2, and tRNALys4 are highly specific for the 5'AAG3' codon. tRNALys5 and tRNALys5a preferentially bind in response to AAA. tRNALys6 binds in response to AAA 3-fold better than in response to AAG. The presence of thiolated nucleosides in the anticodon regions of tRNALys5a, tRNALys5, and tRNALys6 is indicated by I2-inactivation of aminoacylation ability with no effect on the other is isoacceptors. Functional abilities of the isoacceptors were compared in a wheat germ translational system with tobacco mosaic virus RNA as messenger. All of the isoacceptors function about equally well in translation except for tRNALys6, which is only 14 to 24% as effective as the other isoacceptors.

Animals↗
PubMedDate not supplied

Metabolism of nicotine by rat liver cytochromes P-450. Assessment utilizing monoclonal antibodies to nicotine and cotinine.

Because of the prevalence of cigarette smoking in the general population and because studies suggest that a large percentage of nicotine is metabolized to cotinine in humans, it is important to study the enzymes responsible for nicotine metabolism. The cytochromes P-450 have long been implicated in the first step in the conversion of nicotine to nicotine delta 1'(5')-iminium ion. We demonstrate here that rat liver P-450IIB1 is able to convert nicotine to cotinine in the presence of cytosol with a Km of 5-7 microM. A constitutive form of P-450 is also implicated in nicotine metabolism, while purified P-450IA1 and P-450IIC6 show no detectable activity. The lack of P-450IA1 activity substantiates work by others who also failed to observe an increase in the efficiency of nicotine metabolism to cotinine by microsomes from rats that had been pretreated with benzanthracene. This result is in contrast to work with purified rabbit liver enzymes, in which P-450IA1 exhibited low but measurable activity. Our results support the notion that nicotine metabolism to cotinine by P-450 enzymes is highly species dependent. Thus, it is unwise in some cases to extrapolate results obtained by animal model study to the possible role of specific forms of the P-450 enzymes in nicotine metabolism in humans.

Animals↗
PubMedDate not supplied

Interaction of cotinine with rat hepatic microsomal P-450. Comparison with metyrapone and immunomodulation of cotinine and metyrapone binding by monoclonal anti-cotinine antibodies.

The ability of the major nicotine metabolite, cotinine, to interact with rat liver microsomal cytochrome P-450 and the immunomodulatory effects of anti-cotinine antibodies were studied. Cotinine induced type II spectral changes with both microsomes from phenobarbital (PB)-induced rats and purified P-450 with apparent Ks values of 97 and 750 microM, respectively. In contrast, the Ks value was 0.3 microM for metyrapone and 5 microM for nicotine with both the microsomes and purified enzyme. The apparent Ki value for cotinine inhibition of 7-pentoxyresorufin O-dealkylase activity with the microsomes (87 microM) was approximately 87- and 870-fold higher than for nicotine and metyrapone, respectively. Monoclonal antibodies produced against cotinine cross-reacted equally well with metyrapone. They specifically blocked enzyme binding of both drugs based on dose-dependent inhibition of spectral changes, and reversed the metyrapone-induced inhibition of microsomal O-dealkylase activity. In contrast, antibodies to nicotine did not cross-react with cotinine or metyrapone and had no effect on their activity, although they did block the action of nicotine. These results demonstrate that cotinine binding to P-450 from PB-induced rats and inhibition of functional activity in vitro are qualitatively like the effects of metyrapone and nicotine, and that monoclonal anti-cotinine antibodies are useful molecular probes of the interactions between cotinine and metyrapone with the enzyme.

Animals↗