The effects of manually applied intermittent pulsation pressure to rat ventral thorax on lymph transport.

The present study evaluated the effects of tissue massage on a part of the body remote from the region of lymph uptake into the initial lymphatics. Lymph uptake was assessed with a fluorescent probe placed in a potential space of the lower extremity of anesthetized female Sprague-Dawley rats. Tail blood was assayed at intervals over 15 hours for fluorescence. A total of 63 animals were utilized (treatment = 32 and control = 31). The manipulated group received lymph flow enhancing treatment (LFET) five minutes per rat per hour until they were aroused. The control group were left lying prone in cages until a blood sample was taken. The LFET procedure was bilateral finger pressure applied to the lower ribs of a supine rat followed immediately by a light tap to the sternum. These maneuvers were repeated for 5 minutes. The rate of appearance of fluorescent probe was greater during the first nine hours of the experiment in the treatment group than in the controls but not at hours 12 and 15. This study demonstrates that mechanical pressure to body regions physically distant from the location of lymph formation enhances lymph uptake.

Intracellular calcium during photodynamic permeabilization of cardiomyocytes.

The purpose of this study was to determine whether an early increase in [Ca2+]i preceding generalized lysis of cardiomyocytes occurred during photodynamic permeabilization. A method was developed which facilitated the simultaneous measurement, in real time, of permeabilization of the sarcolemma to Ca2+ and Mn2+ during photodynamic action. Quin-2 loaded cells were illuminated in the presence of erythrosin B and the change in the fluorescence emission of the calcium-quin-2 complex was used to measure the rate and extent of change in [Ca2+]i. The same system was used in the presence of extracellular Mn2+ to determine how quickly the cardiomyocytes became permeable to either Mn2+ or quin-2. Calcium ions were observed to enter the myocytes prior to permeabilization of the sarcolemma to either Mn2+ or quin-2, and thus before membrane lysis. Lysis of cardiomyocytes did not appear to be dependent upon increases in [Ca2+]i. Controls were performed to rule out fluorescent artifacts. Reperfusion injury and photodynamic therapy involve both the production of free radicals and an early increase in [Ca2+]i. This study demonstrates a direct correlation between the production of reactive oxygen species and prelytic increases in [Ca2+]i in neonatal cardiomyocytes and demonstrates that this phenomenon may be common to many cell types.

Early increase in intracellular calcium during photodynamic permeabilization.

The purpose of this study was to determine if there was an early increase in intracellular Ca++ which preceded generalized lysis of thymocytes during photodynamic permeabilization. A method was developed that facilitated the simultaneous measurement in real time of permeabilization of the thymocyte cell membrane to Ca++, Mn++, and ethidium bromide during photodynamic action. Quin-2 loaded cells were illuminated in the presence of erythrosin B and the change in the fluorescence emission of the calcium-quin-2 complex was used to determine how soon and to what extent intracellular Ca++ changed following illumination. In the presence of extracellular manganese, the same system was used to determine how soon the cells became permeable to Mn++ or quin-2. It was determined that the fluorescence emission of the ethidium bromide-DNA complex was strong enough to be measured in the presence of the calcium-quin-2 complex. This enabled the concomitant determination of the elapsed time following illumination before ethidium bromide entered the cell. It was established that increased intracellular Ca++ was an early event in the photodynamic permeabilization of thymocytes that preceded permeabilization of the cell membrane to ethidium bromide, Mn++ or quin-2, or lysis.

Permeabilization of thymocytes by photon activation of erythrosin.

Thymocytes previously loaded with quin 2 were rapidly permeabilized by the photon activation of erythrosin and the rate of permeabilization monitored by measuring fluorimetrically the increasing saturation of quin 2 with calcium. The extent of permeabilization was assessed also by the loss of [3H]quin 2 from the thymocytes and penetration of the cells by eosin and trypan blue. Lactate dehydrogenase leakage from the permeabilized cells was markedly delayed compared to the rapid increase in permeability to calcium and quin 2. The rate of permeabilization was dependent upon the concentration of erythrosin, the duration of illumination, the presence of oxygen, and the temperature. These results are consistent with the rapid photochemical generation of highly reactive singlet oxygen which alters thymocyte membrane structure and permeability.

Terbium as a solid-state probe for RNA.

This paper continues previous work on the analysis of nucleic acid-terbium complexes in the solid state. The fluorescence excitation and emission spectra of the RNA-terbium(III) complex is reported. The fluorescence excitation and emission spectra of both the RNA-terbium(III) and DNA-terbium(III) complexes as trapped on millipore filters is reported. One hundred percent of the DNA combined with terbium was trapped on millipore filters. Deoxyribonucleic acid was recovered from DNA-terbium(III) complexes trapped on millipore filters using SDS-extraction. Energy transfer was shown to occur from the bases in nucleic acids to the terbium ion, whereas the actual binding of terbium to nucleic acids was due to phosphate groups. The relative fluorescence of homopolyribonucleotide-terbium complexes showed that the guanine moiety was responsible for most of the observed fluorescence. Binding studies showed an equal affinity of radioactive terbium for all the homopolyribonucleotides. The fluorescence of solid-state DNA and RNA terbium complexes was used to measure picomole quantities of DNA or RNA.

Measurement of binding of terbium to DNA.

This communication demonstrates further that terbium(III) can be used as a probe for DNA. The stoichiometry of terbium binding to DNA was measured by two new methods. In the first method, calf-thymus DNA was titrated with radioactive terbium-160, which is an isotope of the common terbium-159. The resulting DNA-terbium complex was trapped and measured on millipore filters. In the second method, a peak of UV absorption of terbium was found at 219 nm and was used to measure stocichiometry. By both methods, the stoichiometry of binding was one Tb(III) for each three available phosphate groups in DNA. Finally, a rapid method was developed using terbrium-160 to measure the amount of nucleic acid in a solution.

Terbium as a fluorescent probe for DNA and chromatin.

Terbium reacted with DNA and chromatin to form a complex in which terbium acted as a sensitive fluorescent probe. By measuring the narrow-line emission of Tb-3+ when DNA is selectively excited, the relative amount of Tb-3+ bound to the DNA can be calculated. Terbium was bound to DNA until one Tb-3+ was present for each phosphate group. After this point no more terbium was bound. TbCl3 was bound to chromatin in a linear manner until approximately 0.48 TbCl3 was added for each phosphate group in the chromatin-DNA solution. From these data it appears that 52% of the phosphate groups in chromatin were unavailable for binding. The binding of Tb-3+ to DNA can be reversed by prolonged dialysis against 0.5 M NaCl and chelating agents. The terbium ion is ideal in that it binds DNA tight enough so that completion of the reaction can be assumed but loose enough so that it can be removed by gentle means. Low concentrations of salt (up to 2 mM NaCl) enhance the quantum efficiency. Below pH 3 and above pH 7 the DNA-terbium complex will not form. Between pH 3 and pH 7 the quantum efficiency of the DNA terbium complex increases from either pH to a maximum at pH 5.5 to 5.6. Several biochemical uses for Tb-3+ ion are suggested.