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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.