Fluorescent probes for cancer detection.

Several new fluorescent dyes, derivatives of pyrene and of coumarin, were synthesized that have excitation and emission wavelength maxima considerably red-shifted as compared to most pyrene and coumarin compounds. These new fluorescent compounds have high extinction coefficients and high quantum yields, and they also are very environmentally sensitive, which makes them good probes of biological systems. Several of these fluorescent compounds are preferentially taken up and retained by leukemic and other cancer tissue cell lines as compared to normals, particularly 1,3-dihydroxy 6,8-pyrenedisodiumsulfonate and 3-(carboxymethylester)-7-julolidinocoumarin.

Tumor cell specific dark cytotoxicity of light-exposed merocyanine 540: implications for systemic therapy without light.

Merocyanine 540 (MC540) was activated by exposure to 514 nm laser light. The light-exposed MC540 was then mixed (in the dark) with tumor cells and normal cells to determine the antiproliferative activity. Treatment with light-exposed MC540 resulted in 70-90% tumor cell kill from different cell lines, while 85% of the normal human mononuclear cells and 41% of the granulocyte-macrophage colony forming cells (CFU-GM) survived the treatment.

Fluorescent cationic probes of mitochondria. Metrics and mechanism of interaction.

Mitochondria strongly accumulate amphiphilic cations. We report here a study of the association of respiring rat liver mitochondria with several fluorescent cationic dyes from differing structural classes. Using gravimetric and fluorometric analysis of dye partition, we find that dyes and mitochondria interact in three ways: (a) uptake with fluorescence quenching, (b) uptake without change in fluorescence intensity, and (c) lack of uptake.

Increased survival of normal cells during laser photodynamic therapy: implications for ex vivo autologous bone marrow purging.

Laser light-induced, dye-mediated photolysis of leukemic cells was tested in an in vitro model for its efficacy in eliminating occult tumor cells for ex vivo autologous bone marrow purging. Merocyanine 540 (MC540) was mixed with acute promyelocytic leukemia (HL-60) cells in the presence of human albumin. This cell-dye mixture was irradiated with 514 nm argon laser light.

Elimination of leukemic cells by laser photodynamic therapy.

We studied the effects of 514-nm laser light-induced merocyanine 540 (MC540)-mediated toxicity on both leukemic and normal bone marrow (BM) cells. Acute promyelocytic leukemia (HL-60) cells were incubated with MC540 (20 micrograms/ml) and exposed to 93.6 J/cm2 irradiation at a 514-nm wavelength.

McArdle's disease in an elderly woman.

We have described a woman who was 71 years old when McArdle's disease was first diagnosed. She had few symptoms even during severe myoglobinuria. Muscle breakdown occurred upon relatively mild exertion.

Calcium regulates troponin-tropomyosin binding in the reconstituted thin filament.

Steady-state fluorescence anisotropy technique was used to determine the binding constant of troponin for IAEDANS-labeled tropomyosin under various conditions. In the absence of actin, Ca does not affect the binding between troponin and tropomyosin. The presence of actin greatly strengthens troponin-tropomyosin binding in the absence of Ca.

Studies on the spatial arrangement of muscle thin filament proteins using fluorescence energy transfer.

The distance between a pair of fluorophores attached to Cys-36 of beta-tropomyosin and Cys-373 of actin in reconstituted muscle thin filaments was measured by fluorescence energy transfer. Two pairs of donor/acceptor fluorophores, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid/5-iodoacetamidofluorescein and N-(1-pyrene)maleimide/dimethylamino-4-maleimidostilbene, were covalently attached to tropomyosin and actin. The energy transfer efficiencies in various reconstituted systems were determined from decrease in donor lifetime using nanosecond pulse fluorimetry.

Characterization of beta-adrenergic receptor subtypes in androgen-induced mouse kidney hypertrophy using a new high-affinity ligand, [125I]iodocyanopindolol.

In fully developed androgen-induced hypertrophy of female mouse kidney, beta-adrenergic receptors per unit membrane protein were increased approx. 2.5-fold, as measured by the binding of [125I]iodocyanopindolol, with no change in apparent dissociation constants (Kd range 20-25 pM).

Fluorescence spectroscopic studies of pyrene-actin adducts.

Reaction kinetic studies of the sulfhydryl-directed fluorescent probes N-(1-pyrene)maleimide (PM) and N-(1-pyrenyl)iodoacetamide with actin from rabbit skeletal muscle showed that there were three accessible sulfhydryl groups in actin. Fluorescence spectral studies showed energy transfer from aromatic amino acid residues to fluorophore reacted at Cys-373, as well as weak excimer fluorescence probably due to doubly labeled molecules at Cys-10 and Cys-373. These results provide further evidence that trytophan and tyrosine residues are located near the probe attached to Cys-373 or Cys-10 and the latter two thiols are in close proximity.

Increased beta-adrenergic receptor complement in androgen-induced mouse kidney hypertrophy.

Treatment of female mice with testosterone propionate led to a pronounced, but gradual increase in kidney beta-adrenergic receptor complement. The specific binding of [125I]iodohydroxybenzylpindolol rose 2-3-fold above the control levels after 8-12 days of the treatment. No significant changes were detected prior to the fourth day of androgen administration.

Ribosome-membrane interactions: characterization of ribosomal proteins from loose and tight bound ribosomes.

Membrane-bound ribosomes were isolated from a post-mitochondrial supernatant fraction of mouse liver homogenate by sedimentation in a sucrose density gradient, Loose ribosomes were released from the membrane fragments with 0.5 M KCl, while tight bound ribosomes were not released. After purification of the loose and tight ribosomes subclasses, ribosomal subunit proteins were isolated and compared by two-dimensional polyacrylamide gel electrophoresis.

Tissue-specific ribosomal protein composition.

Membrane-bound and free polysomes from murine liver and kidney were isolated under identical conditions and their ribosomal proteins were compared by two-dimensional gel electrophoresis. The results demonstrate that these ribosome subpopulations are quantitatively and qualitatively similar except for the presence of one additional protein in the kidney-bound polysomal fraction.

A general ellipsoid cannot always serve as a model for the rotational diffusion properties of arbitrarily shaped rigid molecules.

An ellipsoidally shaped body, or more commonly, an ellipsoid of revolution, is generally assumed to serve as a convenient model for evaluating the rotational diffusion properties of macromolecules. If Perrin's equations for the rotational diffusion coefficients of general ellipsoids can be shown to generate all possible rotational diffusion coefficients, then there would exist at least one equivalent ellipsoidal shape for every arbitrarily shaped rigid body. We investigated the problem by first generating a space, r-space, representing all possible ellipsoidal shapes.

Subnanosecond single photon counting fluorescence spectroscopy using synchronously pumped tunable dye laser excitation.

A synchronously pumped tunable dye laser has been constructed and interfaced with a modified Ortec 9200 photon counting system for the purpose of measuring subnanosecond relaxation phenomena. The dye laser excitation pulse, which has an intrinsic 35-ps FWHM for Rhodamine 6G, is 350 ps when measured by time-correlated single photon counting. This value appears to be characteristic of the transit time jitter in the RCA 8850 photomultiplier tube.

Dielectric dispersion of actin.

The dielectric dispersion of G-actin was 7 X 10(6) Hz, and the low frequency reduced dielectric increment 0.99 +/- 0.15 ml/mg.

Isolation of renal membranes that contain kallikrein, angiotensin I-converting enzyme (kininase II) and angiotensinase in the rat.

1. Fractions highly enriched in plasma membrane, endoplasmic reticulum or brush border were prepared from rat kidney cortex. Kallikrein was concentrated in the plasma membrane fraction, but not in the brush border fraction.

Isolation of membrane-bound renal enzymes that metabolize kinins and angiotensins.

Cortex of rat kidney was homogenized and fractions enriched in plasma membrane, endoplasmic reticulum or brush border were prepared by several techniques of differential centrifugation. The identity and homogeneity of the membrane fragments were investigated by assaying marker enzymes and by transmission and scanning electron microscopy. Kallikrein was present in both plasma-membrane- and endoplasmic-reticulum-enriched fractions isolated by two fractionation procedures.

Cell-free translation of messenger RNA for a myeloma light chain prepared from synchronised plasmacytoma cells.

Crude preparations of biologically active mRNA, which code for a myeloma (MPC-11) light chain, were isolated by two successive sucrose gradient centrifugations of RNA extracted from membrane-bound ribosomes, mRNA thus obtained was separated into a poly(A)-rich and a poly(A)-poor fraction by oligo(dT)-cellulose chromatography. Both these fractions were able to direct the synthesis of light chains in reconstituted cell-free systems derived from heterologous cells (ascites tumor lysates) and homologous cells (MPC-11 cells grown in suspension culture). The identity of the products in vitro was confirmed by comparing their migration with that of light chains produced in vivo upon electrophoresis in sodium dodecylsulphate/polyacrylamide gels, and from the profiles of tryptic peptides obtained by chromatography on Aminex A-5 ion-exchange columns.