Strand displacement amplification (SDA) and transient-state fluorescence polarization detection of Mycobacterium tuberculosis DNA.

Strand displacement amplification (SDA) is an isothermal, in vitro method of amplifying a DNA sequence for diagnostic purposes. We have combined SDA with fluorescence polarization detection in a closed, homogeneous format. A fluorescently labeled oligodeoxynucleotide detector probe hybridizes to the amplification product that increases in concentration during SDA. The single- to double-stranded conversion of the probe is accompanied by an increase in fluorescence polarization values, which can be measured in real-time without physical manipulation of the sample. The probe was labeled with the near-infrared dye La Jolla Blue, and fluorescence polarization was measured on a transient-state fluorometer. We have applied this homogeneous SDA/detection system to a target DNA sequence specific for Mycobacterium tuberculosis DNA.

Homogeneous detection of nucleic acids by transient-state polarized fluorescence.

We describe a transient-state polarized fluorescence-based method for detecting nucleic acids. An active ester of the phthalocyanine dye La Jolla Blue was coupled to an oligonucleotide containing an amino group at its 5' end, and the conjugate was purified by HPLC chromatography. We monitored the hybridization characteristics of the conjugate with complementary oligonucleotides and RNA as targets by transient-state polarized fluorescence measurements. The method was comparable in sensitivity to isotopic and nonisotopic heterogeneous detection systems and was capable of detecting 1 fmol of a 382-base-long RNA transcript from human immunodeficiency virus type (HIV-1) generated in a self-sustained sequence replication (3SR) reaction.

Doisynolic-type acids--uterotropically potent estrogens which compete poorly with estradiol for cytosolic estradiol receptors.

Doisynolic acids, a class of seco-steroid acids some of which exhibit greater uterotropic estrogenicity than estradiol-17 beta, are D-ring cleavage products of steroidal estrogens formed by fusion with KOH above 200 degrees C. We have found that electron-transfer reactions between estrone or estradiol and CCl4 or CBrCl3 in KOH-t-BuOH at 25 degrees C rapidly provide 16,16-dichloro- or -dibromodoisynolic acid, respectively, the former approaching estradiol in uterotropic potency. Simple esters from these highly hindered tertiary carboxylic acids, easily prepared via phase-transfer-catalyzed alkylations, also rival estradiol in uterotropic activity. Unlike natural steroidal estrogens or their commonly used artificial equivalents (DES, hexoestrol, ethynylestradiol, etc.) whose uterotropic activity is accompanied by substantial binding affinity for cytosolic estradiol receptors, these highly uterotropic doisynolic-type acids and esters exhibit binding affinities for this receptor of only about 1% that of estradiol-17 beta as determined by the usual competitive binding-inhibition studies with [3H]estradiol. Other highly uterotropic carboxylic acids may exhibit similar characteristics. These unusual results leave open the possibilities that uterotropic seco-steroid and related carboxylic acids undergo some unknown metabolic activation, are exceptionally persistent estrogens, bind to a cytosolic receptor site other than the conventional (type I) estradiol site, or bind directly to type I or type II nuclear receptor sites. At dosages of 1000 times those required for a uterotropic effect, the doisynolic-type acids (24 doses over an 8-week period) were neither toxic nor carcinogenic.

Doisynolic acids: potent estrogens with very low affinity for estrogen receptors.

Doisynolic acids are alkaline degradation products of steroidal estrogens. While these doisynolic acids are potent estrogens, some being more estrogenic than estradiol itself, they bind to cytoplasmic estrogen receptors only feebly compared with estradiol.

A fluorescent probe for rapid detection of estrogen receptors.

Fluorescein conjugates to estradiol-17 beta by the sixth carbon (6-FE) and to estrone by the 17th carbon (17-FE) were used to detect estrogen receptors (ERs) in breast cancer tissue sections and in cultured cell lines (human mammary carcinoma MCF-7 and Copenhagen rat prostatic tumor R3327-AT). 17-FE was found to interact with ERs better than 6-FE by biochemical and histochemical techniques. Thin layer chromatography analysis of ethanolic extracts of 17-FE incorporated in tissues and cultured cells showed that over 95% of 17-FE was not metabolized. We concluded that the fluorescence observed in tissue sections and cultured cells was due to 17-FE and not to fluorescein dissociated from the conjugate. Analysis of 65 human breast cancer showed that 74% of the cases were positive for specific 17-FE uptake and 26% were negative. The fluorescence was consistently brighter in the ductal and glandular epithelial cells than in the stroma. specific 17-FE uptake in the nucleoli was observed in MCF-7 and in R3327-AT tumor cells in in vitro cultures, suggesting that these nucleolar estrogen receptors may play a key role in the mechanism of estrogen action. Problems of fluorescence quantitation in tissue sections and cells are discussed.

Estrogen receptor cytochemistry by fluorescent estrogen.

A recently synthesized fluorescein-labeled estrogen (17FE, 1-(N)-fluoresceinyl-estrone-thiosemicarbazone) interacts with estrogen-target cells like the native hormone and visualizes the uptake, transport, and distribution of estrogen in intact target cells. Moreover, estrogen binding sites are traced by 17FE in cryostat sections of estrogen target tissues as well. Cell and tissue 17FE binding sites fulfill the accepted criteria for specific estrogen receptors (finite binding capacity, high affinity, steroid and tissue specificity). This fluorescent probe allows estrogen receptors to be studied in a wide variety of cell and tissue preparations under varying conditions of physiologic and pathophysiologic interest.

Investigation of hormone-receptor interactions by means of fluorescence labeling.

Fluorescent-labeled hormones can be used to study hormone-receptor interactions by means of fluorescence polarization, visualization by fluorescence microscopy, or separation methods, e.g., dextran-coated charcoal. Subcellular fragments, single cells, and tissue preparations are amenable to study; in this work rat uterine cytosol was used unless otherwise noted. Estrone labeled with fluorescein at position 17 gives 50% inhibition in the radiometric dextran-coated charcoal assay at 8.3 X 10(-7) M as compared to 3.4 and 3.5 X 10(-8) M for diethylstilbestrol and estradiol, respectively. Scatchard plots from fluorescence polarization are hyperbolic and consistent with two classes of binding sites having association constants 5.6 X 10(10) and 6.4 X 10(7) M-1. Binding by high-affinity sites, which were present at about 3 times the concentraion of "specific" sites (radiometric dextran-coated charcoal assay), was abrogated by estradiol or diethylstilbestrol. Kinetic measurements showed that binding sites that can be blocked by excess estradiol or diethylstilbestrol are those that are both slowly associating and slowly dissociating. Staining of tissues by estrone labeled with fluorescein at position 17 as seen in the fluorescence microscope showed specificity. In normal rat uterus only epithelial cells were stained. In one human infiltrating ductal carcinoma only the malignant ductoid elements stained, while in another there was essentially no staining.

Sex hormones and the immune response. I. Host factors in the production of penicillin-specific antibodies in the female guinea pig.

Female guinea pigs between 500 and 650 g produced the highest precipitation titers to penicilloyl-coupled guinea pig gamma-globulin of an array of animals ranging in weight from 350 to 850 g. When one or two depot injections in complete Freund's adjuvant were succeeded by saline boosters, the response maximized within 1 week after the first booster (in phase with the maximum output of IgM); subsequent boosters rapidly reduced the titer. The observed response was directed primarily at the hapten, as no reaction was evident to heterologous carrier (KLH) until the anti-hapten titer had declined. The daily administration of estradiol for 23 days after the last antigen inoculation prevented the decay of titer, whereas the titer of the controls or progesterone-treated animals dropped by 40--50% during that interval.

Fluorescein-labeled estradiol: a probe for anti-estradiol antibody.

A fluorescent estradiol derivative binds strongly to antiestradiol antibody. The binding, measured by fluorescence polarization, is inhibited by estradiol and by diethylstilbestrol. Tentatively characterized as N-(estradiol-6-iminooxyacetyl) fluorescein amine, the derivative was prepared from estradiol-6-iminooxyacetic acid, dicyclohexylcarbodiimide, and fluorescein amine, and isolated by TLC. It has a fluorescence emission similar to that of fluorescein and an absorption spectrum consistent with a fluorescein: estradiol molar ratio of 1:1.

Fluorescence polarization measurement of the hormone-binding site interaction.

Fluorescence polarization methodology has been applied to the binding of fluorescent-labeled prolactin, growth hormone and estradiol to subcellular fractions prepared from rabbit mammary and uterine tissue. Equilibrium measurements treated by Scatchard plots have shown that there are high affinity sites (K approximately 10(9) 1 mol-1), as well as lower affinity sites (K approximately 10(8) 1 mol-1) for both hormones. The binding of the fluorescent labeled hormone to microsomal or cytosol fractions has been shown to be inhibited by the prior addition of native, unlabeled hormone. Kinetic results on the interaction of prolactin with the microsomal fraction are consistent with a bimolecular reaction involving significant structural rearrangements during the reaction (not diffusion controlled). The forward rate constant calculated from data on initial rates was found to be 1.7 X 10(5) 1 mol-1 sec-1. Stopped flow kinetic measurements on the reaction between fluorescent-labeled estradiol and cytosol binding sites show that at low temperatures, the reaction goes in two distinct steps separable in time. The second step may be the reaction found by others (utilizing sedimentation velocity methods) which precedes translocation of the hormone-binding site complex to the nucleus. Fluorescence polarization makes it possible to observe both the formation and dissociation of hormone-binding site complexes over a time scale down to a fraction of a second and at concentrations down to the nanogram per nl range.

Isolation of an SV40 induced sarcoma transformation associated antigen.

A transformation associated antigen was isolated from an SV40 induced hamster sarcoma by sequential silica gel column chromatography and preparative silica gel 60 thin layer chromatography after tissue extraction with chloroform:methanol (2:1, v/v). It migrated with an rf of 0.21 on silica gel 60 thin layer chromatography plates predeveloped and developed in chloroform:methanol:water:glacial acetic acid (10:10:1.5:0.5, v/v) and an rf of 0.27 on cellulose F254 thin layer chromatography plates developed in the same solvent system. Antigenicity was determined using a fluorescence probe cytotoxicity assay to measure inhibition of antibody mediated complement dependent damage to homologous cultured transformed cells. Although compositional analysis of this substance is not complete, it appears to be a polar lipid and would support the concept that transformation associated antigens may be gene plus substrate specific rather than strictly gene specific.

Hormone binding by cells and cell fragments as visualized by fluorescence microscopy.

Fluorescent-labeled hormones offer an alternative approach to radio-labeling in studying the binding of hormones to intact cells or cell fragments. The binding of fluorescent-labeled hormones may be followed quantitatively by measurement of the polarization or the binding may be directly visualized in the fluorescence microscope. The binding of both fluorescein labeled prolactin and estradiol to a variety of whole cells or to microsomal fragments has been observed by fluorescence microscopy. No staining was observed with fresh cells whereas all cell types investigated, after freeze-thawing, stained at physiological levels (10-9M) of either hormone. Microsomal preparations from the mammary tissue of mid-pregnant rabbits likewise stained at low levels of prolactin. Inhibition of staining was not produced even by 10-6 M unlabeled hormone.

An improved fluorescence probe cytotoxicity assay.

The phenanthridine dye, ethidium bromide, which is actively excluded by viable cells, undergoes a significant fluorescence enhancement at 5900 A upon binding intracellular double-stranded polyribonucleotides. A rapid and sensitive assay of antibody mediated cytotoxicity to cells grown in vitro has been developed using this phenomenon. In this communication, we describe this fluorescence probe cytotoxicity assay and a sensitive electro-optical system designed to measure the fluorescence enhancement of ethidium bromide as it intercalates with intracellular polyribonucleotides. Basic characteristics of the fluorescence enhancement resulting from the interaction of ethidium bromide and non-viable cells are presented as well as examples of this assay as it has been used to study surface membrane neoantigens of cells tranformed by the oncogenic DNA virus, SV40.

Fluorescence polarization and intensity kinetic studies of antifluorescein antibody obtained at different stages of the immune response.

Kinetic studies of reactions between fluorescein and antifluorescein antibody produced during early, intermediate, and late stages of the immune response have been carried out utilizing both fluorescence intensity and polarization measurements in the static (time constant similar to 5 sec) and in the stopped-flow modes (time constant similar to 5 msec). During maturation of the immune response, it was found that the "on" second-order association rate constant increased its value only by a factor of three, whereas the "off" dissociation first-order rate constant decreased by a factor of over 1000. Hence, it is the rate of dissociation which largely determines the stability of the hapten-antihapten complex. Furthermore, since second-order rate behavior was found for even heterogeneous antibody, most of the heterogeneity with respect to binding affinity occurs as a result of the heterogeneity in the rate of dissociation of the hapten-antihapten complex and not from the primary combination of hapten and antibody. Antifluorescein antibody which exhibits both high binding affinity (K similar to 5 x 10(11) M-1) and homogeneity with respect to equilibrium binding has been shown to obey second-order association kinetics over wide ranges in concentration. Despite the fact that the value of the second-order rate constant for this fluorescein-antifluorescein reaction is as large as that for most other hapten-antihapten reactions (1.4 x 10(8) M-1 sec-1), the binding reaction has an appreciable activation energy (7 kcal/mol). This is true for both divalent and univalent antibody. Furthermore, the reaction rate parameters are markedly affected by specific anions. The value of the second-order rate constant (18.5 degrees) increases according to the following scheme: salicylate less than trichloroacetate less than SCN- less than ClO4- less than Cl- less than F- less than phosphate. The activation energy increases as follows: trichloroacetate less than phosphate less than F- less than Cl- less than ClO4- less than SCN- less than salicylate, whereas estimates of the entropy of activation indicate that deltaS++ increases as follows: tricholroacetate less than phosphate similar to F- less than Cl- less than ClO4- less than SCN- less than salicylate. The same mechanism which was previously proposed by us for the antigen-antibody reaction is also consistent with the kinetics of the fluorescein-antifluorescein reaction. This mechanism postulates a bimolecular process with structural rearrangements (conformational changes and/or the loss of water) in the formation of the transition state complex. The reaction between the fluorescein hapten and its antibody hence is not diffusion limited.

Intracellular supravital stain delocalization as an assay for antibody-dependent complement-mediated cell damage.

The ability of hamster SV40 tumor cells to concentrate Neutral Red into localized intracytoplasmic vacuoles and granules has been correlated with the ability of those cells to adhere and to replicate in tissue culture. Cells damaged by complement-fixing anti-tumor antibody and metabolic inhibitors first undergo delocalization of the dye, which appears as a diffuse stain throughout the nucleus and cytoplasm. More severely damaged cells lose the stain entirely, at a stage of progressive cell damage correlated with Trypan Blue uptake. A rapid and sensitive cytotoxic assay procedure has been based upon Neutral Red staining behavior, and the assay has been used to study antibody-dependent, complement-mediated cytotoxic anti-SV40 tumor activity in the sera of normal hamsters.