texas-red and tetramethylrhodamine-isothiocyanate

[corrected] Prostate-specific membrane antigen (PSMA) remains an attractive target for imaging and therapeutic applications for prostate cancer. Recent efforts have been made to conjugate inhibitors of PSMA with imaging agents. Compared to antibodies, small-molecule inhibitors of PSMA possess apparent advantages for in vivo applications. To date, there are no reports on the cellular fate of such constructs once bound the extracellular domain of PSMA. The present study was focused on precisely defining the binding specificity, time-dependent internalization, cellular localization, and retention of inhibitor conjugates targeted to PSMA on LNCaP cells. A novel fluorescent inhibitor was prepared as a model to examine these processes.. Fluorescence microscopy of LNCaP and PC-3 cell lines was used to monitor the specificity, time-dependent internalization, cellular localization, and retention of a fluorescent PSMA inhibitor.. Fluorescent inhibitor 2 was found to be a potent inhibitor (IC50 = 0.35 nM) of purified PSMA. Its high affinity for PSMA on living cells was confirmed by antibody blocking and competitive binding experiments. Specificity for LNCaP cells was demonstrated as no labeling by 2 was observed for negative control PC-3 cells. Internalization of 2 by viable LNCaP cells was detected after 30 min incubation at 37 degrees C, followed by accumulation in the perinuclear endosomes. It was noted that internalized fluorescent inhibitor can be retained within endosomes for up to 150 min without loss of signal.. Our results suggest that potent, small-molecule inhibitors of PSMA can be utilized as carriers for targeted delivery for prostate cancer for future imaging and therapeutic applications.

Topics: Amides; Antigens, Surface; Binding, Competitive; Cell Line, Tumor; Fluorescent Dyes; Glutamate Carboxypeptidase II; Humans; Inhibitory Concentration 50; Male; Microscopy, Fluorescence; Phosphoric Acids; Prostatic Neoplasms; Rhodamines; Xanthenes

Topics: Analog-Digital Conversion; Animals; Color; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Lasers; Microscopy, Fluorescence; Proteins; Rhodamines; Software; Subcellular Fractions; Subtraction Technique; Xanthenes

Four porcine sperm plasma membrane proteins were previously identified as putative ligands for the oocyte plasma membrane. The present study examined the binding of these proteins and two additional porcine sperm membrane proteins to oocytes from sheep, mice and hamsters as a first step in assessing potential conservation of these putative sperm ligands across species and across mammalian orders. Plasma membrane vesicles were isolated from porcine sperm, solubilised, and the proteins separated by one-dimensional gel electrophoresis. The 7, 27, 39 and 62 kDa porcine sperm protein bands demonstrating predominant binding of the porcine oocyte plasma membrane on ligand blots, a 90 kDa protein band demonstrating minor binding, and a 97 kDa protein band that did not bind the oocyte plasma membrane probe were electroeluted. Proteins were biotinylated, and incubated with zona-free oocytes. Bound biotinylated protein was labelled with fluorescent avidin and the oocytes examined with a confocal microscope. The 7 kDa, 27 kDa and the 39 kDa proteins bound to the sheep oocytes but not to a majority of the hamster or mouse oocytes. The 62 kDa protein bound to sheep oocytes and mouse oocytes but not to a majority of the hamster oocytes. The 90 kDa protein bound to oocytes from all three species. The 97 kDa protein, which did not recognise the porcine oocyte probe on a Western ligand blot, did not bind to oocytes from any species and served as a negative control. These observations are consistent with significant conservation of molecule and function among species within the same mammalian order. Hence, one species may be a good model for other species from the same order. Only limited conservation of binding activity of porcine sperm plasma membrane proteins to rodent oocytes was observed, suggesting a greater divergence either in molecular structure or in function among species from different orders.

Topics: Animals; Avidin; Cell Membrane; Cricetinae; Female; Fluorescent Dyes; Male; Membrane Proteins; Mice; Molecular Weight; Oocytes; Rhodamines; Sheep; Species Specificity; Spermatozoa; Swine; Xanthenes

We present a new method that combines protein-specific fluorescence staining with confocal microscopy to simultaneously measure matrix and surface protein deposits via optical sectioning of hydrogel contact lenses. Tetramethylrhodamine isothiocyanate (TRITC), Texas Red, fluorescein isothiocyanate (FITC), and fluorescein succinate were used to selectively react with the lysine residues of protein deposits for two lens types (etafilcon A [a 58% water, ionic lens] and polymacon [a 38% water, nonionic lens]). Because TRITC and Texas Red gave high levels of nonspecific staining, they were discontinued. Following exhaustive rinsing of lenses, central lens buttons were analyzed using a Bio-Rad MRC-500 laser scanning confocal microscope. Optical sections were made every 4 microns at 200x magnification, and the fluorescence signals were processed using image analysis software. The high water ionic material accumulated protein much more rapidly than the low water nonionic material. For a given lens type a correlation was observed between the wear time and the degree of protein deposition; however, we did observe significant inter-subject variations in total protein. From this preliminary work, we conclude that this confocal method is feasible and desirable for simultaneous determination of surface and matrix protein deposition.

Topics: Contact Lenses, Hydrophilic; Eye Proteins; Fluorescein-5-isothiocyanate; Humans; Hydrogel, Polyethylene Glycol Dimethacrylate; Microscopy, Confocal; Polyethylene Glycols; Rhodamines; Xanthenes

A method fo nonradioactive DNA sequencing, which has been marketed commercially, uses four different fluorescent tags to label DNA fragments and fixed-wavelength excitation/fluorescence detection of the labels. This study presents an alternative method of detection based on synchronous luminescence (SL), in which both excitation and emission wavelengths are scanned simultaneously. This approach has proven in the past to have significant advantages over fixed-wavelength luminescence in the analysis and identification of fluorescent analytes. In this paper, the utility of synchronous excitation was investigated as a method for DNA sequencing with fluorescent tags. A laser-based SL instrument, recently developed in this laboratory, was used to resolve the spectra of a mixture of the dyes used in the fluorescence-based sequencing scheme. A preliminary limit of detection of 720 zeptomoles (10(-21) M) of fluorescein isothiocyanate dye in solution was achieved with this instrument. The results presented here suggest that the SL technique could result in an increase in sensitivity and a decrease in error rate of identification during DNA sequencing.

Topics: 4-Chloro-7-nitrobenzofurazan; Cytidine Monophosphate; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Hydrogen-Ion Concentration; Luminescent Measurements; Rhodamines; Sequence Analysis, DNA; Spectrometry, Fluorescence; Xanthenes

Topics: Animals; Fluorescent Dyes; Histocytochemistry; Kidney; Rabbits; Rhodamines; Wheat Germ Agglutinins; Xanthenes